COFFEE CONSTITUENTS AND MODULATION OF
ANTIOXIDANT STATUS IN CACO 2 CELLS
by
YAZHENG LIU
B. Sc., Shandong University, 2006
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES (Food Science)
THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)
March 2010
© Yazheng Liu 2010 ABSTRACT
Coffee contains biologically active components which may affect chronic disease risk.
These biologically active components include caffeine, cafestol and kahweol, and antioxidants such as chlorogenic acids and Maillard reaction products (MRPs) that are generated during roasting. Although MRPs are regarded as being the most abundant group of antioxidants present in coffee, the mechanism underlying the antioxidant effects of coffee MRPs in both in vitro and in biological systems has yet to be elucidated.
In this study, the in vitro antioxidant properties of roasted and non roasted coffee extracts
(Coffea arabica L.) were tested using oxygen radical absorbance capacity (ORAC),
Trolox equivalent antioxidant capacity (TEAC) and reducing power assays. MRPs were shown to be the prevailing antioxidants in roasted coffee extracts. The mechanisms of the antioxidant action associated with coffee MRPs involve the hydrogen atom transfer (HAT) mechanism and the single electron transfer (SET) mechanism.
The biological effects of MRPs derived from coffee extracts on the enzymatic antioxidant defense in human colon adenocarcinoma Caco 2 cells were also investigated. No induction of antioxidant enzyme activities of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase were observed in Caco 2 cells after exposure to coffee MRPs, except for an increased glutathione peroxidase activity after 24 h exposure.
In contrast, significantly decreased activities of catalase and glutathione peroxidase, and a reduced glutathione content were observed in Caco 2 cells after treatment with coffee
MRPs (p<0.05).
ii The antioxidant gene expression profile in Caco 2 cells after coffee treatment was further investigated using a Real Time Polymerase Chain Reaction (PCR) array technology.
Results demonstrated that roasted coffee extracts induced the expression of specific antioxidant response element (ARE) driven genes in Caco 2 cells, thus enhancing cellular endogenous defense systems. This is the first report of the molecular mechanism underlying the antioxidant effect of coffee in Caco 2 cells. Hydrogen peroxide generated in the cell culture system as a consequence of coffee exposure, may serve as a signaling molecule that is involved in the gene regulatory effect associated with coffee extracts.
iii TABLE OF CONTENTS
ABSTRACT...... ii
TABLE OF CONTENTS...... iv
LIST OF TABLES...... viii
LIST OF FIGURES ...... x
LIST OF ABBREVIATIONS...... xiii
ACKNOWLEDGEMENTS...... xvi
CHAPTER I
OVERVIEW: GENERAL INTRODUCTION, LITERATURE REVIEW, AND
RESEARCH HYPOTHESES AND OBJECTIVES...... 1
1.1 GENERAL INTRODUCTION...... 2
1.2 LITERATURE REVIEW ...... 6
1.2.1 Oxidative stress and antioxidants...... 6
1.2.1.1 Reactive oxygen species and oxidative stress...... 6
1.2.1.2 Antioxidant mechanisms...... 7
1.2.1.3 Gene regulations by oxidative stress...... 7
1.2.2 Maillard Reaction (MR)...... 10
1.2.2.1 Chemistry of Maillard Reaction (MR)...... 10
1.2.2.2 Chemistry of Maillard reaction products (MRPs) ...... 11
1.2.2.3 Antioxidant properties of MRPs ...... 14
1.2.2.4 MRPs and chemoprotective enzymes ...... 17
1.2.3 Coffee – a source of MRPs ...... 18
1.2.3.1 Composition of coffee bioactive components...... 19
iv 1.2.3.2 Coffee as a source of dietary antioxidant...... 25
1.2.3.3 Coffee consumption and health ...... 29
1.3 RESEARCH HYPOTHESES AND OBJECTIVES...... 32
CHAPTER II
CHEMICAL CHARACTERISTERCS AND ANTIOXIDANT PROPERTIES OF
COFFEE...... 35
2.1 Introduction...... 36
2.2 Materials and methods ...... 38
2.2.1 Coffee preparation ...... 38
2.2.2 Ultrafiltration ...... 38
2.2.3 Physical chemical analyses ...... 39
2.2.4 Chemical based antioxidant assays...... 40
2.2.5 Cell based assays...... 42
2.2.6 Statistical analysis...... 47
2. 3 Results...... 48
2. 3.1 Yields and recovery of coffee brews and ultrafiltration fractions ...... 48
2.3.2 Chemical characteristics of coffee...... 50
2.3.3 Antioxidant activity of coffee extracts in chemical systems ...... 55
2.3.4 Biological effects of coffee extracts ...... 60
2.4 Discussion...... 66
2.4.1 Chemical characteristics of coffee...... 66
2.4.2 Antioxidant activity of coffee extracts...... 68
2.4.3 Biological effects of coffee extracts ...... 70
v CHAPTER III
COFFEE CONSTITUENTS AND MODULATION OF OXIDATIVE STATUS IN
CACO 2 CELLS...... 72
3.1 Introduction...... 73
3.2 Materials and method...... 75
3.2.1 Preparation of coffee and Maillard reaction products (MRPs)...... 75
3.2.2 Chemical analyses and Antioxidant assays...... 76
3.2.3 Cellular in vitro Assay ...... 77
3.2.4 Real Time Quantitative Reverse Transcription PCR (RQ RT PCR) Array .... 78
3.2.5 Statistical analysis...... 81
3.3 Results...... 82
3.3.1 Recovery of coffee brews and fractions...... 82
3.3.2 Chemical characteristics of coffee...... 83
3.3.3 Antioxidant activity of roasted and green coffee...... 88
3.3.4 Biological effects of coffee bean extracts...... 91
3.3.5 Biological effects of coffee fractions on Caco 2 cells ...... 99
3.3.6 The regulatory effects of coffee on the expression of the genes involved in the
oxidative stress and antioxidant defense system in Caco 2 cells...... 105
3.3.7 Chemical characteristics and antioxidant activity of Suc Ser and Ara Ser
model MRPs...... 109
3.3.8 Chemical characteristics and antioxidant activity Ara Ser model MRPs
fractions...... 117
3.3.10 Cell based bioactivity of Ara Ser MRPs ...... 121
vi 3.3.11 Biological effects of Ara Ser MRPs fractions on Caco 2 cells ...... 128
3.3.12 Gene regulation of MRPs on the human oxidative stress and antioxidant
defense system (HOSAD) in Caco 2 cells...... 132
3.4 Discussion...... 135
3.4.1 Chemical characteristics of coffee extracts and model MRPs...... 135
3.4.2 Antioxidant activity and reducing power of coffee constituents ...... 138
3.4.2 Biological effects of green coffee, roasted coffee and model MRPs on the
antioxidant defense system in Caco 2 cells ...... 143
3.4.3 Coffee and the expression of Redox sensitive genes in Caco 2 cells...... 147
CHAPTER IV
GENERAL DISCUSSION AND CONCLUSIONS...... 155
4.1 GENERAL DISCUSSION ...... 156
4.1.1 Chemical characteristics and antioxidant properties of coffee ...... 156
4.1.2 Coffee, antioxidant enzymes and antioxidant genes...... 157
4.2 CONCLUSION...... 159
4.3 SUGGESTIONS FOR FUTURE RESEARCH...... 161
REFERENCES ...... 162
APPENDIX...... 185
vii LIST OF TABLES
Table 1.1 Example of antioxidant defense systems……………………………………….9
Table 1.2 Composition of green and roasted coffee……………………………………..19
Table 1.3 Caffeine content of different coffee beverages…………………….…………20
Table 1.5 Summary of potential health benefits of coffee consumption from
epidemiological studies…………………………………….…….…………...31
Table 2.1 Recovery yields of coffee extracts………………………………………….49
Table 2.2 Recovery of coffee fractions by water and salt ultrafiltrations………………..49
Table 2.3 Color parameters (L, E) and browning of fractionated coffee extracts…..51
Table 2.4 Antioxidant activities of coffee extracts and fractions……………………..…57
Table 2.5 Antioxidant activity of defatted non fractionated coffee extracts and
recombined extracts…………………………………………………..….…..58
Table 2.6 Antioxidant activities of coffee fractions by water and salt ultrafiltration……59
Table 2.7 IC 50 of coffee extracts on Caco 2 cells using MTT assay……...……………..62
Table 3.1 Human oxidative stress and antioxidant defense PCR array gene table ……...79
Table 3.2 Recovery of coffee fractions by ultrafiltration……………………..….………82
Table 3.3 Lightness (L) and browning of coffee extracts and untrafiltration fraction.…83
Table 3.4 Antioxidant activity of coffee extracts…………………..……………………89
Table 3.5 Antioxidant activity of coffee fractions determined by the ORAC method….90
viii Table 3.6 Antioxidant activity of coffee fractions determined by the TEAC method…..91
Table 3.7 Antioxidant activity of coffee fractions determined by the RP method……91
Table 3.9 IC 50 of coffee extracts on Caco 2 cells using MTT assay…………………….92
Table 3.10 Caco 2 MTT response to coffee with and without H 2O2 treatment………101
Table 3.11 Genes differently expressed in Caco 2 cells after incubation with coffee
extracts and hydrogen peroxide...……………………………………….…107
Table 3.12 Lightness (L) and browning of model MRPs………………..…..…….111
Table 3.13 Antioxidant activity of Suc Ser and Ara Ser MRPs……………..……….116
Table 3.14 Recovery of Ara Ser MRPs ultrafiltration fractions………………………..117
Table 3.15 Lightness (L) and browning of fractions derived from Ara Ser model MR
system……………………………….…………………..……………....….118
Table 3.16 Antioxidant activity of fractionated Ara Ser MRPs…………….………….121
Table 3.17 IC 50 values of Ara Ser MRP extracts on Caco 2 cells using MTT assay….124
Table 3.18 Caco 2 MTT response to MRPs with (+) and without ( ) H 2O2 treatment…128
Table 3.19 Genes differently expressed in Caco 2 after incubation with Ara Ser MRP.133
ix LIST OF FIGURES
Figure 1.1 CGA degradation during roasting of coffee bean………………..……...……22
Figure 1.2 Contribution of coffee to the antioxidant intake in diet……………………....26
Figure 1.3 Mechanism of scavenging of free radicals by caffeine………………...…….29
Figure 2.1 Fluorescence emission spectra (350 550 nm) of light roasted and dark roasted
coffee extracts and fractions.……………………………...……………..….53
Figure 2.2 Comparison of the UV visible spectra of light roasted and dark roasted coffee
extracts and fractions…….…………………………………………….……54
Figure 2.3 Effects of light roasted and dark roasted coffee extracts on the tetrazolium
reduction rate in the MTT assay…………………….……………..………..61
Figure 2.4 Effect of coffee extracts on catalase (CAT) activity in Caco 2 cells………...63
Figure 2.5 Effect of coffee extracts on glutathione (GSH) content in Caco 2 cells……..65
Figure 3.1 Fluorescence emission spectra (350 550 nm) of green bean, light roasted and
dark roasted coffee extracts and fractions.………………………..…….…..87
Figure 3.2 Comparison of the UV visible spectra of green bean, light roasted and dark
roasted coffee extracts and fractions……………………………….….…....88
Figure 3.3 Alpha dicarbonyl compounds in light roasted and dark roasted coffee
extracts………………………….………………………………….….…....89
Figure 3.4 Effects of green bean, dark roasted and light roasted coffee extracts on the
tetrazolium reduction rate in the MTT assay……………………...……..….94
Figure 3.5 Effect of coffee extracts on glutathione (GSH) content in Caco 2 cells……..95
Figure 3.6 Effect of coffee extracts on glutathione peroxidase (GPX) activity in Caco 2
cells……………………….…………………………………………….…...97
x Figure 3.7 Effect of coffee extracts on catalase (CAT) activity in Caco 2 cells….…….98
Figure 3.8 Effect of light roasted and green bean coffee extracts on Caco 2 cellular GSH
contents with and without H 2O2 treatment……………….....……………..101
Figure 3.9 Effect of light roasted and green bean coffee extracts on Caco 2 cellular
antioxidant enzyme activities with and without H 2O2 treatment……….….104
Figure 3.10 Antioxidant genes expression in Caco 2 cells treated with light roasted, dark
roasted coffee extracts and H 2O2 compared to those in control cells.…..…108
Figure 3.11 Fluorescence emission spectra (350 550 nm) of light roasted and dark roasted
Sugar Serine MRPs extracts………………...………………………..……111
Figure 3.12 UV spectra of light roasted and dark roasted Sugar Serine MRPs extract...111
Figure 3.13 Alpha dicarbonyl compounds in Ara Ser MRPs and Suc Ser MRPs crude
extracts…………………………………….……………………………….115
Figure 3.14 Fluorescence emission spectra (350 550 nm) of light roasted and dark roasted
MRPs extracts and fractions……………………….………………………119
Figure 3.15 Comparison of the UV spectra of light roasted and dark roasted MRPs
extracts and fractions………………………………………………………120
Figure 3.16 Effects of light roasted and dark roasted) Ara Ser MRPs extracts on the
tetrazolium reduction rate in the MTT assay………………………………123
Figure 3.17 Effect of Ara Ser MRPs extracts on glutathione (GSH) content in Caco 2
cells...... 125
Figure 3.18 Effect of Ara Ser MRPs extracts on glutathione peroxidase (GPX) activity in
Caco 2 cells……………………………………………………………...…126
xi Figure 3.19 Effect of Ara Ser MRPs extracts on superoxide dismutase (SOD) activity in
Caco 2 cells……………………………………………………….………..127
Figure 3.20 Effect of MRPs extracts and associated fractions derived from light roasted
Ara Ser MR system on Caco 2 cellular glutathione (GSH) contents after 24 h
of treatment……………………………………………………….…..……129
Figure 3.21 Effect of light roasted (LR) Ara Ser MRP extracts and fractions on Caco 2
cellular antioxidant enzyme activities after 24 h of treatment…...……..….130
Figure 3.22 Antioxidant genes expression in Caco 2 cells treated with light roasted and
dark roasted Ara Ser MRPs compared to those in control cells……….…..134
Figure 4.1 UV vis spectrum of a typical melanoidin and of individual chromophoric sub
structures…………..……………………………………………..……...…137
Figure 4.2 Chlorogenic acids and related compounds according to chemical
characteristics…………………………………………………………..…..140
Figure 4.3 Proposed pathway of H 2O2 formation in coffee brews……………………...154
xii LIST OF ABBREVIATIONS
AAPH 2,2’ azobis(2 amidinopropane) dihydrochloride
ABTS 2,2’ azinobis(3 ethylbenzothiazoline 6 sulfonate)
AP 1 Activator protein 1
Ara Arabinose
ARE Antioxidant response element
BHA Butylated hydroxyanisole
BHT Butylated hydroxytoluene
C+K Cafestol and kahweol
Caco 2 Human intestinal adenocarcinoma cancer cell line
CAT Catalase
CCR NADPH cytochrome c reductase
CFAQ Caffeoylferuloyl quinic acids
CGA Chlorogenic acid
CQA Caffeoylquinic acid
DR Dark roasted
GB Green coffee beans
GPX Glutathione peroxidase
GR Glutathione reductase
GSH Glutathione
GST Glutathione S transferase h Hours
HAT Hydrogen atom transfer
xiii HepG2 Human liver carcinoma cell line
HMF Hydroxymethyl furfural
HPLC High performance liquid chromatography iNOS Inducible nitric oxide synthase
Int 407 Human embryonic intestinal cell line
Keap1 Kelch like ECH associated protein 1
LR Light roasted
MEM Minimum essential medium
MR Maillard reaction
MRPs Maillard reaction products
MTT 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide
MW Molecular weight
NF κB Nuclear factor kappa B
Nrf2 Nuclear factor erythroid 2 p45 subunit related factor 2
• O Superoxide radicals 2
ONOO Peroxynitrite radical
ORAC Oxygen radical absorbance capacity
PBS Phosphate buffer saline pCoA p coumaric acid
PCR Polymerase chain reaction
PR Reducing power
• RO Peroxyl radicals 2
xiv ROS Reactive oxygen species
SD Standard deviation
Ser Serine
SET Single electron transfer
SOD Superoxide dismutase
Suc Sucrose t BOOH tert butylhydroperoxide
TE Trolox equivalents
TEAC Trolox equivalent antioxidant capacity
xv ACKNOWLEDGEMENTS
My graduate study was a journey with my supervisor and committee, my parents, my grandparents, lab colleagues, graduate secretaries and many friends from UBC Bible study group. They are the people who make this journey colorful, enjoyable and unforgettable, and finally to fruition.
My supervisor, Dr. David Kitts, has a special way to keep me going. He told jokes to encourage me when I fell down, and treated me for coffee when I accomplished even little things. He was always willing to help me, and he never pushed me too much. He said: when you are happy, I am happy. I would like to thank Dr. Kitts for his care, guidance, patience and trust. Especially, I am very thankful for the countless hours he has spend helping me with the scientific writing. Dr. Scaman always has a smile on her face when she saw me. I can feel the encouragement and care through the smile. I appreciate her time to answer my questions regarding to the statistical analysis, and especially thank her for the concern and understanding of the progress of my graduate study. I want to give special thanks to Dr. Adams for allowing me to use the equipment in his lab. I also want to thank him for making the time to my meetings and replying my emails very quick.
There were sunny days and rainy days during the journey. However, no matter what kind of day, my grandma and my parents were with me and supported me with all they could.
Because of them, I was able to run through the whole journey. Thank you very much for your prayers! Grandma! Thank you very much! Dad! Mom! I love you! I am heartily thankful to my grandpa, Don Smith, and grandma, Elaine Smith, whose encouragement, love and support from the beginning to the last, enabled me to stay in this foreign country with joy and happiness. My grandma is a good listener and advisor. I shared many things
xvi with her, things that I was happy and unhappy with. I would like to thank grandma for listening to my problems and always staying on my side. I would like to extend great thanks to Shaowei Dong for his constant love and support, and those delicious dishes that he made for me. I would also like to give special thanks to Andrea Goldson, who helped me with research problems, revise my writings, and took me to wonderful events and activities, which are all good memories of my graduate study. She did so much for me. I would like to give my deepest thanks to Andrea. I am also appreciative of Fanchui Gang, who taught me how to use Endnote and shared his knowledge of chemistry. I am grateful for working in Dr. Kitts’ lab with an excellent team of graduate students and lab technicians. I would like to thank Ingrid Elisia, Xiumin Chen, Alexandra Tijerina Saenz,
Monica Purnama, Steve Tomiuk, Minh Huynh and Katie Hu for teaching me techniques, helping me with research problems and giving me guidance for graduation. Also, I would like to thank Kirsten Cameron, Lia Maria Dragan, Allison Barnes, Val Skura and Pedro
Aloise for their help, support and hard work. This thesis would not have been possible without the prayers from UBC Bible study group. I would like to give sincere thanks for their prayers. Lastly, I would like to give my regards and blessings to all of those who supported me in any respect during the completion of my project.
xvii
CHAPTER I
OVERVIEW: GENERAL INTRODUCTION, LITERATURE REVIEW, AND
RESEARCH HYPOTHESES AND OBJECTIVES
1 1.1 GENERAL INTRODUCTION
The role of food and its components on human health continues to be a keen topic of
interest to both the lay public and academics. The potential of forming beneficial or
harmful products in food as a result of food processing is a major area of investigation by
food toxicologists. One particular reaction that has received continuing interest for more
than 100 years is the Maillard, or browning reaction. This reaction takes place during the
thermal processing of food and involves the condensation of amino groups from amines,
amino acids, peptides, or proteins with carbonyl groups from sugars or fatty acids
(Maillard, 1912; Hodge, 1953). The reaction develops into a complex network of
chemical intermediate products, including fluorescent, color and flavor compounds, and polymerized brown end products, all herein referred to as Maillard reaction products
(MRPs) (Hodge, 1953). MRPs can be divided into two classes; namely the low molecular
weight colored compounds that consist of four linked ring structures, with molecular
weights below 1 KDa, and the high molecular weight MRPs, which are colored polyphenolic structured compounds (Arnoldi et al. , 1997; Hofmann, 1997, 1998a; Ames et al. , 1999a). The high molecular weight MRPs, termed melanoidins, have a molecular weight up to 300 KDa (Ibarz et al. , 2009).
Besides the sensory properties attributed to MRPs, some negative aspects of this reaction
also exist, including the destruction of essential amino acids, a decrease in digestibility of proteins, and the potential production of products with carcinogenic, mutagenic and toxic potential; all of which can have an impact on both the quality and safety of heated foods
(OBrien and Morrissey, 1989). On the other hand, however, some MRPs have been
2 demonstrated to have health promoting properties. Melanoidins, for example, recently
have been shown to possess antioxidant activity (Wijewickreme and Kitts, 1997, 1998a;
Delgado Andrade et al. , 2005), antimicrobial activity (Rufian Henares and Morales,
2007a), antihypertensive activity (Rufian Henares and Morales, 2007c), chemopreventive and antimutagenic properties (Powrie et al. , 1986; Faist et al. , 2001; Somoza et al. , 2003),
as well as prebiotic effects (Ames et al. , 1999b).
MRPs are an abundant group of compounds that also exist in coffee brews and therefore
represent a significant part of the diet for those that consume coffee beverages. High
molecular weight melanoidins (MW>10KDa) derived from coffee have antioxidant properties that involve metal chelating of prooxidants (Wijewickreme and Kitts, 1998b;
Morales et al. , 2005), free radical scavenging of peroxyl radicals and hydroxyl radicals
(Morales and Jimenez Perez, 2004; Morales, 2005), and other free radicals (Borrelli et al. ,
2002). In line with the chemical antioxidant activity, coffee melanoidins have also been
shown to protect human hepatoma HepG2 cells against oxidative damage induced by
tert butylhydroperoxide (Goya et al. , 2007). However, a dose dependent reduction of
glutathione (GSH) content in HepG2 cells was also observed after 24 h coffee
melanoidins treatment (Goya et al. , 2007). The mechanism underlying this observation is
unknown. The question remains as to whether MRPs that show antioxidant activity in
chemical assays, will also function as antioxidants in biological systems.
Relatively little research has been dedicated to study the antioxidant properties of low
molecular weight MRPs (MW<1KDa), derived from coffee brews. One study found that
low molecular weight coffee components (MW<1KDa) possess higher antioxidant
activity compared to the high molecular weight components (Somoza et al. , 2003), which
3 indicates that the low molecular weight MRPs derived from coffee may have higher antioxidant activity compared to the high molecular weight melanoidins. It is still unclear
if there is a relationship between the antioxidant activity of MRPs and the related
molecular weights.
Caco 2 cells derived from human colon adenocarcinoma cells are widely used to model
the small intestine for investigating the effects of food components on cell function and
metabolism. These cells are often used to examine the biological response to an oxidative
stress and mitigation by nutritional antioxidants (Baker and Baker, 1993; Cepinskas et al. ,
1994; Manna et al. , 1997; Wijeratne et al. , 2005). The activity and genetic expression of
antioxidant enzymes in Caco 2 cells have been reported to change with time after
confluence (Baker and Baker, 1992). Therefore, both homogeneously undifferentiated (at
subconfluence) and 100% confluence Caco 2 cells were used in this thesis to study the
oxidative stress responses induced by both coffee and model MRPs.
This research thesis is composed of two experiments. Experiment I attempted to
characterize and compare the antioxidant properties of light roasted and dark roasted
coffee, and related fractions that have different molecular weights. The biological effects
of coffee extracts on the cellular antioxidant status in Caco 2 cells were also investigated.
The objectives for Experiment II were to evaluate the antioxidant activity of MRPs
derived from coffee brews and to further explore the underlying antioxidant mechanisms
associated with coffee MRPs. The biological effects of MRPs derived from coffee on the
cellular antioxidant defense in Caco 2 cells were also investigated. Finally, human
oxidative stress and antioxidant defense PCR array was used to gain an understanding of
changes in the antioxidant gene expression in Caco 2 cells after coffee extract treatment.
4 This experiment provides an insight into the mechanisms of antioxidant status modulatory effects associated with coffee constituents at the molecular level.
5 1.2 LITERATURE REVIEW
1.2.1 Oxidative stress and antioxidants
1.2.1.1 Reactive oxygen species and oxidative stress
Oxygen is an essential requirement for normal growth and metabolism of the body.
However, related products of cellular respiration, often referred to as reactive oxygen species (ROS), possess a tremendous potential for toxicity which is manifested by
oxidation of important cellular constituents that can eventually result in cell death. ROS
are generated endogenously by autoxidation and through cellular metabolic reactions that involve both mitochondria and peroxisomes, not to mention, many cytosolic enzymes and
non enzymatic systems (Sies, 1993). In addition, exposure to exogenous factors, such as
ultraviolet light, radiation, chemotherapeutics, smoking and some specific environmental toxins will also trigger the production of ROS (Leanderson and Tagesson, 1990; Sies,
1993). ROS attack lipids, exciting chain reactions that can cause cumulative oxidative damage. Since many studies indicate ROS to be an underlying cause for ageing, chronic disease and death (Raha and Robinson, 2000), there is a continuous requirement for the inactivation of ROS within the body. High doses and/or inadequate removal of ROS will result in oxidative stress (Sies and Cadenas, 1985), a condition which has been proposed to initiate mutagenesis, carcinogenesis and cardiovascular disease (Waris and Ahsan,
2006; Valko et al. , 2007).
6 1.2.1.2 Antioxidant mechanisms
The exposure of cells and organ systems to a high partial pressure oxygen environment
will result in oxidative stress. Survival from a hyperbaric state is possible through the
action of strategically located enzymatic and non enzymatic antioxidants, and by the continued replacement and repair of oxidative damaged tissue macromolecules. The term
“antioxidant” can therefore be used to describe any substance that delays or inhibits oxidative reactions, albeit the ultimate effectiveness on removing oxidative stress will differ (Kitts, 1997).
1.2.1.2.1 Non enzymatic and enzymatic antioxidants
A cellular antioxidant defense system consists of a collective function of non enzymatic and enzymatic antioxidants that work in concert to reduce the potential toxicity of ROS.
Table 1.1 provides an overview of some antioxidants that have received interest from scientists and characterizes specific antioxidant defense mechanisms (Sies, 1993; Yuan and Kitts, 1997). These include: (1) the scavenging of free radicals and singlet oxygen
(e.g. vitamin E and superoxide dismutase), (2) the reduction of hydroperoxides (e.g. glutathione peroxidase and catalase), (3) the removal of metal catalysts (prooxidants) from the site of action (e.g. proteins and chelating agents).
1.2.1.3 Gene regulations by oxidative stress
The ability of cells to cope with, or prevent, the damage caused by oxidative stress is an
important component of cellular, and by extension whole body homeostasis. In human
cells, gene expression is modulated by ROS (Valko et al. , 2007). This modulation has been observed in response to both direct and indirect oxidative challenge and involves
7 changes at many levels that include transcription, mRNA stability, and signal
transduction (Crawford et al. , 1988; Devary et al. , 1991; Wang et al. , 1996; Hartsfield et al. , 1997). Numerous specific genes related to the enzymatic antioxidant defense system have been identified. These include genes that encode glutathione peroxidases (GPX), peroxiredoxins (PRDX), superoxide dismutases (SOD), and oxidative stress responsive genes, not to mention other genes that are involved in ROS metabolism. Modest inductions by oxidative stress of enzymatic antioxidant mechanisms, such as SOD, GPX, or catalase (CAT), have been observed in mouse muscle cells (Franco et al. , 1999).
Studies have found that several protooncogenes, for example, c fos , c myc, and c jun , were induced by ROS, such as hydrogen peroxide (Crawford et al. , 1988; Devary et al. ,
1991; Nose et al. , 1991). These genes are critical to cellular growth and differentiation and an onset of an aberrant expression may potentially result in cancer (Zheng and
Hendry, 1997). Oxidant/antioxidant responsive elements (ARE) have been identified in the promoter region of several genes, which include glutathione S transferase (GST) Ya subunit, c fos , c jun , heme oxygenase and NAD(P)H:quinine oxidoreductase (Rushmore and Pickett, 1993; Venugopal and Jaiswal, 1998). These genes are stimulated at the transcriptional level by hydrogen peroxide. Reports have identified an element in the
GST Ya subunit that is bound and activated by a series of antioxidant compounds
(Nguyen et al. , 1994). Interestingly, the affinity to stimulate transcription by antioxidants is based on how well they produce critical levels of ROS (Crawford, 2002). Several important transcriptional factors have been identified that are mediators of oxidative stress (Hancock et al. , 2001). These factors are induced, or activated, by ROS and then bind and activate those genes that are involved in the overall cellular antioxidant defense
8 systems. Most notably, these include nuclear factor kappa B (NF κB) and activator protein 1 (AP 1). These genes are also excellent biomarkers for assessing cellular antioxidant defense systems, and represent valuable potential targets in the treatment of oxidative stress related diseases.
Table 1.1 Example of antioxidant defense systems (Sies, 1993; Yuan and Kitts, 1997) System Functionality Non enzymatic Albumin binds Fe, Cu ions Ascorbate (vitamin C) electron donor , singlet oxygen quencher, regenerate α tocopherol radical Bilirubin plasma antioxidant Flavonoids plant antioxidants Glutathione (GSH) thiol group maintain redox potential Lycopene electron donor , singlet oxygen quencher Ubiquinol 10 radical scavenger Urate radical scavenger Uric acid free iron binding α tocopherol (vitamin E) radical chain breaking: electron donor, hydrogen donor, free radical scavenger, singlet oxygen quencher β carotene electron donor , singlet oxygen quencher Enzymatic (direct) Catalase (CAT) mainly located in cellular peroxisomes and to some in the cytosol; catalyzes the reduction of hydrogen peroxide. GSH peroxidases (GPX) plasma, intracellular. Reduce hydrogen peroxide and lipid peroxides to water and lipid alcohols. Superoxide dismutases (SOD) plasma, milk, cytosol, mitochondria. Contain redox metals in the catalytic center and convert dismutase superoxide radicals to hydrogen peroxide and oxygen. Enzymatic (ancillary enzymes) Conjugation enzymes glutathione s transferase, UDP glucuronosyl transferases: conjugates xenobiotics and alkylating agents with GSH GSSG reductase maintain GSH levels NADPH supply NADPH for GSSG reductase NADPH quinoe oxidoreductase two electron reduction Repair systems DNA repair systems oxidized protein turnover oxidized phospholipid turnover Transport systems GSSG export Thioether (s conjugate) export
9 1.2.2 Maillard Reaction (MR)
The Maillard reaction (MR) occurs quickly during heating and was first reported by L.C.
Maillard in 1912. It is a complex series of non enzymatic reactions that involves free
amino groups reacting with carbonyl groups that result in a browning reaction. MR is one
example of a non enzymatic browning reaction that is very important in many food
systems and produces desirable attributes such as flavour, texture and color of food.
Many intermediate products with bioactive properties are also generated during the
Maillard reaction. These may include potential carcinogens, mutagens, antimutagens,
antioxidants, allergens and antiallergens (Friedman, 2005), albeit considerable
inconsistency in the scientific literature exists.
1.2.2.1 Chemistry of Maillard Reaction (MR)
The complete chemical description of the MR is yet to be fully defined. The earliest
systematic review of the reaction scheme was put forward by Hodge in 1953, and further
modified by Reynolds in 1969 and Mauron in 1981. Basically, the reaction was divided
into three stages which are:
(1) The initial stage, which consists of sugar amine condensation and Amadori or Heyns
rearrangement, forming a Schiff base.
(2) The intermediate stage, which consists of sugar dehydration and fragmentation, as
well as amino acid degradation. Many low molecular weight intermediate compounds are produced during this stage. For example, highly reactive α amino carbonyl compounds
are formed by Strecker degradation, the third reaction in the formation of MRPs. In this
reaction, condensation of these intermediate compounds produces heterocyclic
10 compounds, which contribute to many flavours in heated food, such as coffee. Some
fluorescent compounds and brown pigments also occur, but at very low concentrations at
these particular stage.
(3) The final stage of the MR is a polymerization reaction that produces high molecular
weight, colored end products. These are referred to specifically, as melanoidins in food systems and advanced glycation end products (AGEs) in body tissues.
1.2.2.2 Chemistry of Maillard reaction products (MRPs)
Maillard reaction products (MRPs) consist of a vast number of reaction products. In general, low molecular weight MRPs are very important in flavor and off flavor production, while high molecular weight MRPs/melanoidins are the ultimate end products of the reaction. Attempts to summarize the proposed structure of melanoidins
was made by Goya et al. (Goya et al. , 2007) and included different end products, such as:
(i) Low molecular weight colored substances that crosslink with free amino groups of
lysine or arginine in proteins; (ii) units of furan and/or pyrroles that react through polycondesation reactions to form melanoidin repeating units; (iii) the melanoidin
chemical skeleton, which is mainly built up from sugar degradation products formed in
the early stages of the reaction, and often polymerized and cross linked by amino
compounds. Since the composition of MRPs is greatly influenced by several factors, such
as the ratio and type of amino compounds, together with the presence of reducing sugars, pH, temperature, time, and water activity (Wijewickreme and Kitts, 1997), it is expected
that the final composition of MRPs will reflect the complex reactions that are involved in
the multiple chemical schemes describing a variety of potential products.
11 Spectrophometric measurements are commonly used to characterize and quantitate the
generation of complex MRPs. Broad spectral peaks occurring between 250 nm to 350 nm
are often associated with low molecular weight MRPs (Jing and Kitts, 2003). Some
compounds derived from early stage MRPs generation, including pyrazines and
hydroxymethyl furfural (HMF) compounds, are detected near the maximum absorbance
at 280 nm (Lerici et al. , 1990; Ames et al. , 1999a). The alkaline degradation of reducing
sugars leads to the generation of chromophores at both 210 nm and 265 nm. Maximum
absorption at 265 nm has been attributed to the presence of α dicarbonyl intermediates
(Jing and Kitts, 2003). Polymerization of the intermediates occurs at the late stages of the
MR with the formation of melanoidins that are detectable in the visible region. A single
wavelength measurement of brown pigments at 420 nm is frequently used to measure the
rate and extent of the final stage of the Maillard reaction (Morales and Jimenez Perez,
2004).
The color parameters provided by tri stimulus colorimetry are also commonly applied to
indicate the visual color attributed to non enzymatic browning (Morales and van Boekel,
1998; Morales and Jimenez Perez, 2001). Chemical analyses of the brown pigments (or buff orange) has confirmed the presence of furans, pyrroles, and pyridines (Rizzi, 1997).
Pyrazine formation was related to color formation, and as pyrazine products increased the
color of the MRPs changed from colorless to yellow, then to brown and finally to darker brown pigments (Wong and Shibamoto, 1996). Morales and Jimenez Perez (Morales and
Jimenez Perez, 2001), examined the heating of model MRPs at 100 ºC for 24 h that
included glucose alanine (GA), glucose glycine (GG), glucose lysine (GL), lactose
alanine (LA), glucose glycine (LG), and lactose lysine (LL) and recorded changes in the
12 L, a, b tri stimulus coordinates. The lightness indicator L decreased significantly during
heating in these model systems, which indicates increased darkness at the final stage of
the Maillard reaction. A net increase in a yellow brown color was observed during the first hours of heating before reaching a maximum. This was followed by a color change to orange brown, even purplish red for GL and LL MRP samples at higher heating times.
GL and LL systems produced the darkest color. High concentrations of sugars in the open chain form have been found to brown faster and more intensely (Boekel, 2001). Color formation is therefore due to both the presence of low molecular weight and the high molecular weight MRPs (Ames, 1992). Other workers suggested that the redness parameter, a, may be a reliable indicator of acrylamide levels, which are generated under very specific MR conditions and represent harmful intermediates in certain thermally processed foods, such as fried potatoes (Gokmen and Senyuva, 2006).
While the development of color is an important feature of the MR, some studies have placed emphasis on the generation of fluorescent MRPs. It has been suggested that
different MRPs with fluorescent properties are related to increased heating conditions,
such as at prolonged heating (Morales and Jimenez Perez, 2001). Some fluorescent
MRPs are involved in the formation of colored MRPs and may be the precursors of brown pigments (Leclere and Birlouez Aragon, 2001). The fluorescent compounds
mentioned above did not follow the same time dependent trend as colored compounds in
defining the MR model systems (Morales and Jimenez Perez, 2001). Fluorescent
molecules are stable with prolonged heating, whereas complexes that are brown will
change almost linearly with the duration of heating time (Morales and Jimenez Perez,
2001).
13 1.2.2.3 Antioxidant properties of MRPs
MPRs, especially melanoidins present in processed food and generated in model systems
have been intensively studied in recent years. Evidence for an antioxidant role for MRPs is supported by many in vitro and some in vivo studies (Mastrocola and Munari, 2000;
Faist and Erbersdobler, 2002; Delgado Andrade et al. , 2005; Kitts and Hu, 2005;
Michalska et al. , 2008). Heating glucose with amino acids has resulted in a remarkable
scavenging activity towards hydroxyl radical (Kawane et al. , 1999). Several heterocyclic
MRPs, which are major flavour compounds, show antioxidant activity by inhibiting
hexanal oxidation and lipid peroxidation, and scavenging thyrosyl radicals (Macku and
Shibamoto, 1991). Melanoidins from the glucose glycine model system exhibited
antioxidant properties by quenching ROS (Wagner et al. , 2002). It has also been reported that melanoidins prepared from a xylose glycine model system have antioxidant activity comparable to BHA and BHT (Hayase et al. , 1999). Furthermore, MRPs from a glucose
tryptophan model system can exhibit a synergistic effect with tocopherol in inhibiting
lipid autoxidation (Chiu et al. , 1991). Studies indicate that the antioxidant activity of
MRPs is depended on the type of sugar (Wijewickreme and Kitts, 1997; Sun et al. , 2006;
Chen and Kitts, 2008b). It has been shown that the configuration of OH group on carbon
moieties C 3 and C 4 are important for the formation of MRPs and related antioxidant
activities (Sun et al. , 2006). MRPs can also exert prooxidant activities in some cases. For
example, glucose lysine model MRPs generate free radicals in the presence of trace
amounts of iron, which in turn causes the degradation of hyaluronan (Deguine et al. ,
1998). Other studies have shown that the fructose lysine model MRPs exhibit more
14 prooxidant and genotoxic activities compare to glucose lysine model MRPs in the presence of copper ions (Wijewickreme and Kitts, 1997).
The radical scavenging activity of MRPs can also progressively increase with the
intensity of heat treatment and the development of browning (Murakami et al. , 2002).
The antioxidant property of MRPs therefore occurs to some extent in the later stage of the
MR, or from the generation of melanoidins. However, other studies have reported that browning cannot be directly related to the free radical scavenging properties of MRPs
formed over prolonged heating conditions (Morales and Jimenez Perez, 2001; Jing and
Kitts, 2002). For example, in some cases, fluorescence measurement of heated MR
system is correlated better with free radical scavenging activities (Morales and Jimenez
Perez, 2001). In MR model systems, where the development of melanoidins was the final
outcome, antioxidant activity of MRPs derived from the same sugar or same amino acid
model systems, was inversely related to the fluorescent intensity (Chen and Kitts, 2008b).
Some studies have also found that the high molecular weight MRPs, which contribute to
the color pigments, also show antioxidant activities (Monti et al. , 1999), while others
found the antioxidant activity occurred mainly within the intermediate and low molecular
weight MRPs (Nienaber and Eichner, 1995). It can be deduced that color and fluorescent properties of MRPs are useful indicators of the different stages of the Maillard reaction
and can be used as indicators of high molecular weight and low molecular weight MRPs
formation, but may not absolutely reflect antioxidant capacity potential.
The chemical antioxidant property of MRPs is well accepted and has important
applications to the food industry. However, the effects of MRPs on the antioxidant
enzyme activity have not always been found to be desirable. Former studies have
15 reported that glutathione reductase (GR) and catalase (CAT) activities, and glutathione
(GSH) content in human lymphocytes were decreased when exposed to MRPs derived
from different sugar lysine model systems (Yen et al. , 2002). The activities of GR, CAT
and ascorbate peroxidase (APX) decreased in mung bean seeds in proportion to the
increase of MRPs during storage (Murthy et al. , 2002). Sugar lysine MRPs inhibited the
antioxidant enzyme activity of superoxide dismutase (SOD), CAT, and glutathione peroxidase (GPX), and the total GSH content in human intestinal epithelial Caco 2 cells, while sugar casein model MRPs decreased SOD, GPX, GR activities in Int 407 cells and had no effect on Caco 2 cells (Jing and Kitts, 2004b). A study showed that the dicarbonyl compound, methylglyoxal, generated during the early stage of the Maillard reaction, can
inhibit GPX activity by binding to GSH binding sites (Park et al. , 2003). Feeding mice
methylglyoxal significantly decreased liver SOD, glutathione S transferase (GST), CAT,
glyoxalase I and II antioxidant enzyme activities and was associated with a decrease in
GSH content, along with an increase in lipid peroxidation. It was suggested that
methylglyoxal generates free radicals, which in turn lowers the antioxidant status in
animals (Ueda et al. , 1998). Finally, one study found that a MRP rich diet, which had
antioxidant activity in vitro , had no effect on modifying the oxidative status in healthy
humans (Seiquer et al. , 2008). There is very little in vivo data to indicate an antioxidant
defense mechanism for MRPs in animals, and human intervention trials (Kitts et al. ,
1993). Thus the question remains whether dietary intake of these compounds can exert an
antioxidant effect in the human body beyond that of the gastrointestinal tract.
16 1.2.2.4 MRPs and chemoprotective enzymes
MRPs can also be recognized as xenobiotics; a term which classifies compounds that are
not formed endogenously and require detoxifying mechanisms to protect the organism
from harmful effects (Somoza, 2005). These detoxifying mechanisms collectively
contribute to a chemopreventive potential. Most chemopreventive, non endogenously formed agents act by modulating the Phase I carcinogen activating enzymes and Phase II detoxifying enzymes. Phase I metabolic transformations include reduction, oxidation, and hydrolytic reactions, while Phase II transformations act through conjugation reactions of the xenobiotics, or on Phase I metabolites. A decrease in Phase I enzyme activity and associated increase in Phase II enzyme activity are considered events that have the most effective chemopreventive potential (Somoza, 2005).
Phase I NADPH cytochrome c reductase (CCR) and phase II GST in Caco 2 cells have been shown to decrease after incubation with low molecular weight (<10 KDa) or high molecular weight (>10KDa) glucose glysine melanoidins (Hofmann et al. , 2001). Similar
results were found for a glucose casein model system, where GST activity in Caco 2 cells
was decreased after exposure to nondialysed glucose/casein melanoidins (Faist, 2001).
These results also showed that the effects on CCR and GST were mediated by both high
molecular weight and low molecular weight MRPs. However, low molecular weight
compounds were more effective than high molecular weight compounds (Hofmann et al. ,
2001). Also, methyglyoxal, isolated from the low molecular weight MRPs fraction and
tested on Caco 2 cells, was shown to increase the activity of CCR and decrease the
activity of GST (Hofmann et al. , 2001). In an animal study, mice fed a diet containing glucose lysine MRPs exhibited a significant decrease of Phase I hydrocarbon
17 hydroxylase (AHH) and Phase II UDP glucuronyltransferase (UDP GT) activities in the
small intestine mucosa (Kitts et al. , 1993). Generally, the chemopreventive effect of modeled MRPs in these studies was not overly promising. However, further studies were also carried out in order to see the effects of food derived MRPs on Phase I and Phase II enzymes. The chemopreventive action of bread crust on cultured Caco 2 cells resulted in an induction of GST and reduced CCR (Lindenmeier et al. , 2002). These results were
confirmed by animal feeding studies. When bread crust was fed to rats at a moderate,
human diet equivalent intake for 15 days, the activities of Phase II GST and UDP GT in
the liver increased, and the total antioxidant capacity in the plasma was also enhanced
(Somoza et al. , 2005). These studies also demonstrated that pronylated amino acids and proteins, as part of melanoidins can act as antioxidant and chemopreventive agents in
vitro and in vivo . Another chemopreventive compound formed during heat treatment was
identified in roasted coffee, where pronylated amino acids and proteins seemed to be present at very low amounts. N methylpridinium was shown to have strong
chemopreventive effects on modulating Phase II enzymes both in vitro and in vivo
(Somoza et al. , 2003).
1.2.3 Coffee – a source of MRPs
Coffee is one of the most popular beverages consumed in the world, and is known for its
desirable taste and aroma, stimulant effects and many potential health related benefits.
Recently, an increased number of papers have been published on various potential health benefits of coffee consumption (Tavani and La Vecchia, 2004; Ranheim and Halvorsen,
2005; van Dam and Hu, 2005; Cadden et al. , 2007; Gomez Ruiz et al. , 2008). Many
18 studies have shown that coffee consumption is associated with a reduced risk of several
chronic diseases (Giovannucci, 1998; Tavani and La Vecchia, 2004; van Dam and Hu,
2005; Barranco Quintana et al. , 2007; Cadden et al. , 2007; Larsson and Wolk, 2007).
Various physiological and pathological responses can be attributed to the bioactive compounds, including caffeine, chlorogenic acids (CGA), Maillard reaction products
(MRPs) and diterpenes kahweol and cafestol (K+C). Table 1.2 reports the overall chemical composition of green beans and roasted coffee beans (Arya and Rao, 2007).
Table 1.2 Composition of green and roasted coffee (adopted from (Arya and Rao, 2007)) Constituent Green (%DB) a Roasted (%DB) b Hemicellulose 23.0 24.0 Cellulose 12.7 13.2 Protein (non alkaloid N) 11.6 3.1 Fat 11.4 11.9 Chlorogenic acids 7.6 3.5 Sucrose 7.3 0.3 Lignin 5.6 5.8 Caffeine 1.2 1.3 Trigonelline 1.1 0.7 Reducing sugars 0.7 0.5 Unknown 14.0 31.7 Total 100.0 100.0 a Dry Green Beans. b not corrected for dry weight roasting loss, which varies from 2 5%.
1.2.3.1 Composition of coffee bioactive components
1.2.3.1.1 Caffeine
Caffeine content in coffee beverages can be quite variable, depending on the type and source of coffee beans, roasting method and how the coffee is prepared (Barone and
Roberts, 1996; Harland, 2000; McCusker et al. , 2003). Robusta beans have a relatively
higher caffeine content than Arabica (Charrier, 1975). Dark roasted coffee contains less
19 caffeine than coffee made from light and medium roasted beans (Anon, 2004). The
caffeine content of coffee has shown to vary significantly between brands and the day to day serving frequency (McCusker et al. , 2003). See Table 1.3.
Table 1.3 Caffeine content of different coffee beverages Coffee Caffeine (mg) in 8 oz serving Brewed 135 Ground roasted, percolated 118 Ground roasted, drip 180 Instant 106 Starbucks espresso 280 Starbucks, mocha, latte, Americano 35 Starbucks regular 130 Maxwell House regular 110 Big Bean regular 82 Data from (Barone and Roberts, 1996; Harland, 2000; McCusker et al. , 2003)
Caffeine is absorbed in the stomach and small intestine and metabolized primarily in the
liver (McCusker et al. , 2003). It is almost completely absorbed and distributed to all tissues, including the brain due to its relatively small size and optimal hydrophobic character. The plasma half life of caffeine ranges from 2.3 to 12 h, depending on the physiological or health condition of the individual (Baselt, 2002). Peak caffeine plasma concentrations occur at 45 min to 2 h after ingestion (Ellenhorn and Barceloux, 1988).
Caffeine appears to exert most of the biological effects through the antagonism of the potent endogenous neuromodulator, adenosine (Dunwiddie and Masino, 2001). The effect of caffeine is generally stimulatory, including central nervous system stimulation, acute elevation of blood pressure, increased metabolic rate, and diuresis (Carrillo and
Benitez, 2000).
20 1.2.3.1.2 Chlorogenic acids (CGA)
Chlorogenic acids (CGA) are abundant phenolic compounds present in coffee, with
caffeoylquinic (CQA), feruloylquinic (FQA), and dicaffeoylquinic (diCQA) acids being the major subclasses. Depending on the coffee bean cultivar, green coffee beans contain between 6 14 % CGA on a dry matter basis (Farah and Donangelo, 2006). It has been
shown that the CGA concentration in Arabica green bean extracts varied between 16.6 %
to 22.4 % (w/w) due to different extraction procedures (with water) (Budryn et al. , 2009).
During roasting CGA undergoes a progressive destruction and transformation (Figure 1.1)
(George et al. , 2008). Nevertheless, coffee beverages are still a major dietary source of
CGA. In roasted Arabica coffee extracts, CGA ranged from 2.6 % to 15.8 % (w/w),
depending on the roasting degree and extraction methods (e.g. water) (Budryn et al. ,
2009). It has been estimated that one cup (240 ml, 8 oz) of ground roasted Arabica coffee
contains 80 230 mg CGA, compared to 80 400 mg in a cup (240 ml, 8 oz) of Robusta
coffee. Instant coffee can provide between 35 110 mg CGA per gram of soluble powder
(Farah and Donangelo, 2006).
A recent study indicated that all major CGA in coffee are bioavailable and are absorbed
and/or metabolized differently in humans (Monteiro et al. , 2007). They reported that
there are two major temporal absorption patterns of CGA after coffee consumption,
which suggested an early absorption in the stomach followed by absorption throughout
the small intestine (Monteiro et al. , 2007). The C max (maximum plasma concentration) of total CGA was found to vary from 4.7 to 11.8 mol/L, among six individual human subjects and T max (time corresponding to C max ) for total CGA varied significantly between individuals (from 1 to 4 h) (Monteiro et al. , 2007). Chlorogenic acids were also
21 shown to be metabolized by the liver and gut microflora into various aromatic acid
metabolites (Gonthier et al. , 2003; Mateos et al. , 2006). Enterohepatic circulation of
CGA has been observed for up to 48 h after phenolic intake, suggesting that CGA
undergoes a gradual utilization and excretion in humans (Cremin et al. , 2001). The biological properties of dietary CGA will depend on the whole body kinetic flux of the phenolic acid which in turn involves absorption, metabolism, distribution and interaction
with target tissues (Cremin et al. , 2001; Monteiro et al. , 2007).
Chlorogenic acid
+ Quinic acid Caffeic acid Slow Degradation Rapid Degradation
+ + + +
Catechol 4-ethyi Catechol Hydro quinone Catechol Pyrogallol Gallic acid
Figure 1.1 CGA degradation during roasting of coffee bean (George et al. , 2008)
1.2.3.1.3 Maillard reaction prducts (MRPs) in coffee
During the roasting process of coffee, carbohydrates and protein are degraded and the
Maillard reaction that occurs leads to the formation of flavour and colored products
(Oosterveld et al. , 2003). These MRPs are responsible for the development of the
characteristic brown color and the basic taste of bitterness and astringency common to
22 coffee. The MRPs that are regarded as important contributors to the coffee flavour are the volatile aroma compounds (Yanagimoto et al. , 2002). During roasting, phenolics, especially CGA are partially degraded and bound to MRP polymer structures, thus contributing to some extent to the brown Maillard products (Delgado Andrade et al. ,
2005; Bekedam et al. , 2008a; Bekedam et al. , 2008b). Studies have shown that melanoidins make up 25 % (w/w) of coffee dry matter (Borrelli et al. , 2002), and the concentration increases with increased roasting time (Sacchetti et al. , 2009). It has been shown that arabinogalactan is the most abundant sugar present in the melanoidin rich coffee fractions, and that the residual amount of this sugar is affected by roasting, with a consequent loss of arabinose (DeMaria et al. , 1996; Bekedam et al. , 2008a). Researchers
(Oosterveld et al. , 2003) showed that coffee polysaccharides are degraded during roasting and may be involved in MRP formation. In addition, studies have shown that amino acids present in coffee beans are degraded during roasting, and that the nitrogen from these amino acids may end up in melanoidin structures (Macrea, 1985; DeMaria et al. , 1996).
Arginine, lysine, serine, threonine, histidine and asparagine have also been shown to be significantly reduced during the roasting process of coffee beans (Macrea, 1985;
Bekedam et al. , 2006). Some researchers found that serine was the most affected amino acid during the roasting process, which also was suggested to be an important flavour precursor in coffee (DeMaria et al. , 1996). Coffee proteins, especially those that contain highly reactive ε amino, thiol, or methylthiol groups, undergo chemical changes upon roasting and are likely to be involved in melanoidin formation (Rizzi, 1999; Bekedam et al. , 2006; Bekedam et al. , 2007).
23 Some products in the initial stage of the Maillard reaction (e.g. Amadori rearrangement product, ARP) are degraded via different pathways (Ames, 1992; Anese and Nicoli,
2003), thus, leading to the formation of reductones and furfurals. In vivo , these products are absorbed by diffusion and metabolized by the colonic microbiota (Erbersdobler and
Faist, 2001). Most metabolic transit data on melanoidins has been obtained in rats (Faist and Erbersdobler, 2001). Generally, melanoidins, of any source are characterized as having a low digestibility and bioavailability (Borrelli and Fogliano, 2005), with the relatively small fraction absorbed being speculated to be utilized to low degree since they are excreted in the urine in a slightly modified or unmodified form (Erbersdobler and
Faist, 2001).
1.2.3.1.4 Cafestol and kahweol (C+K)
Ditepene cafestol and kahweol (C+K) represent the major part of the unsaponifiable lipid fraction present in coffee beans. Commercial ground roasted coffees contain about 1%
(w/w) of diterpenes (Urgert et al. , 1995). These diterpenes comprise up to 10 15% of the lipid fraction of roasted coffee beans (Lercker et al. , 1995). The brewing method is a major determinant of diterpene content in coffee beverages (Urgert et al. , 1995; Gross et al. , 1997). Diterpenes are extracted from ground coffee during brewing, but are mostly removed by paper filters. Turkish coffee, Boiled, and French press brews contain relatively high levels of C+K, while filtered, percolated, and instant coffee contain low levels of C+K (Urgert et al. , 1995; Gross et al. , 1997). Studies performed in ileostomy patients indicate that about 70% of the C+K in unfiltered coffee is absorbed intestinally
(De Roos et al. , 1998). Only a small part of the diterpenes is excreted in urine, which
24 indicated an extensive metabolism of C+K in human body (Ratnayake et al. , 1993;
Urgert et al. , 1996).
1.2.3.2 Coffee as a source of dietary antioxidant
1.2.3.2.1 Antioxidant intake in human diet
Coffee has been reported to have high antioxidant activity, which may be of great benefit in improving the quality of life of consumers by preventing, or postponing, the onset of many age related degenerative diseases. Researchers showed that coffee contained the greatest antioxidant potential among 34 common beverages (Pellegrini et al. , 2003). The antioxidant activity of coffee beverages was over six times greater than that of green tea, and about three times as high as that found in red wines. It is particularly noteworthy that coffee represents a major source of dietary antioxidant intake in Germany (Radtke et al. ,
1998), Spain (Pulido et al. , 2003), the United Kingdom (Clifford, 1999), and Norway
(Svilaas et al. , 2004) (Figure 1.2). Although the antioxidant properties of coffee have been attributed to caffeine, the formation of MRPs during roasting, and the relatively
great extent a number of phenolic compounds, likely supersedes the presence of caffeine
in terms of contributions to total antioxidant activity (Daglia et al. , 2000; del Castillo et al. , 2002; Caemmerer and Kroh, 2006).
25
A B
Figure 1.2 Contribution of coffee to the antioxidant intake in diet. A. Norway (Svilaas et al. , 2004); B. Spain (Pulido et al. , 2003).
1.2.3.2.2 Antioxidant property of phenolics in vitro
Chlorogenic acids are the predominant phenolics found in green coffee beans, which
contribute to most of the overall antioxidant activity (Caemmerer and Kroh, 2006). The
free CGA content in roasted coffee is lower than that in the green coffee bean due to the
degradation of CGA at thermal roasting temperatures. However, recent research has
demonstrated that the antioxidant activity of CGA was not completely destroyed despite
the chemical alterations that occur with heating (Bekedam et al. , 2008c). In other words,
CGA does not totally lose the phenolic nature, albeit, the active moiety that provides
antioxidant activity is not retained as free CGA. Instead, CGA degradation likely
involves a complex interaction where it is bound to other molecules via ionic and ester bonds (Delgado Andrade et al. , 2005; Nunes and Coimbra, 2007; Bekedam et al. , 2008d).
Phenolic antioxidants therefore may still contribute to the overall antioxidant activity of
coffee beverages, but in a transformed state. In addition to the direct scavenging effect by
26 CGA on ROS and free radicals, which explain the affinity to inhibit the oxidation and peroxidation to low density lipoprotein (LDL) (Castelluccio et al. , 1995) and decreased
ROS induced DNA damage (Yamanaka et al. , 1997), other studies have shown that CGA
can up regulate some cellular xenobiotic phase II enzymes (Kitts and Wijewickreme,
1994; Feng et al. , 2005) and suppress ROS mediated NF κB, AP 1, and mitogen
activated protein kinase (MAPK) activation (Feng et al. , 2005).
1.2.3.2.3 Coffee MRPs and the antioxidant potential
Many heterocyclic compounds derived from the Maillard reaction have been identified
and quantified in coffee brews, including pyrroles, oxazoles, furans, thiazoles, thiophenes,
imidazoles, and pyrazines (Fuster et al. , 2000). These compounds all possess antioxidant
activity, with pyrroles showing the highest activity relative to thiazoles and pyrazines
having the least activity in inhibiting hexanal oxidation (Fuster et al. , 2000; Yanagimoto
et al. , 2002). Underlying mechanisms for this apparent activity has been suggested to be
facilitated by the electron density of the carbon atoms present on the heterocyclic ring,
and different functional groups on the heterocyclic ring (Eiserich and Shibamoto, 1994;
Yanagimoto et al. , 2002). Under mild roasting conditions, CGA has been shown to be the
main component responsible for the free radical scavenging activity of coffee brews (del
Castillo et al. , 2002). However, MRPs may also be the principal component with free
radical scavenging activity in more severely roasted coffees (del Castillo et al. , 2005).
Some researchers (Borrelli et al. , 2002) found that the antiradical activity of coffee
melanoidins decreased as the intensity of roasting increased, but the affinity to prevent
linoleic acid peroxidation was higher in the dark roasted coffee samples. Pretreatment of
human HepG2 cells with digested coffee melanoidins prevented the increase in cell
27 damage evoked by tert butylhydroperoxide (Goya et al. , 2007). Researchers suggested that the antioxidant activity of coffee melanoidins could be attributed to the incorporated
CGA and CGA degradation products (Delgado Andrade and Morales, 2005; Delgado
Andrade et al. , 2005). This incorporation may also enable CGA in the human colon to interact with gut microbiota, which plays an important role in maintaining health (Tuohy et al. , 2003). Moreover, new antioxidative structures formed through the Maillard reaction are also present in melaniodins (Nicoli et al. , 1997; Bekedam et al. , 2008c). Low molecular compounds released from coffee melanoidins after gastrointestinal digestion can exert antioxidant activity when assayed by five different methods, and the antioxidant activity was even higher than melanoidins and compounds ionically bound to melanoidins (Rufian Henares and Morales, 2007b). Volatile coffee MRPs were proposed to possess potential antioxidant activity by preventing DNA damage in vitro
(Wijewickreme and Kitts, 1998c) and influence gene expression in rats brain (Seo et al. ,
2008).
1.2.3.2.4 Caffeine contributes to the antioxidant activity of coffee
Caffeine and metabolites exhibit both antioxidant and prooxidant properties in vivo , which depend on many parameters such as dose, level of atmospheric O 2 exposure, presence of transition metals, and the biological and chemical end points used for the
measurement of activity (George et al. , 2008). Caffeine is an effective inhibitor of lipid peroxidation as shown by the experiment where millimolar concentrations of caffeine
scavenged ROS (Devasagayam et al. , 1996). At physiological concentrations, caffeine
metabolites can prevent LDL oxidation (Lee, 2000). In general, the antioxidant ability of
caffeine was shown to be similar to that of the antioxidant glutathione (GSH), and
28 significantly higher than that of ascorbic acid (Devasagayam et al. , 1996). The
antioxidant activity of caffeine and its metabolites was probably attributed to the presence
of the carbonyl group at the C8 position of the pyridine ring (George et al. , 2008) (Figure
1.3). It is suggested that this chemical structure enables caffeine to scavenge highly
reactive free radicals, such as hydroxyl radical (OH•), and the generated caffeine radical
may be excreted in urine or stabilized by other antioxidants.
+
Figure 1.3 Mechanism of scavenging of free radicals by caffeine (George et al. , 2008).
1.2.3.3 Coffee consumption and health
To support many of the biological/biochemical activities attributed to coffee components,
it is noteworthy that many studies have shown that coffee consumption is associated with
reduced risk of several chronic diseases. Caffeine is the most widely studied coffee
component, however, it is not the major contributor to many beneficial health related
effects (Levin, 1982; Corrao et al. , 2001; Greer et al. , 2001). Table 1.4 summarizes the
quantitative assessments of the relationship between coffee consumption and the risk of
several diseases from a meta analysis of epidemiologic studies. A systematic review of 9 prospective cohort studies, including more than 193,000 men and women, found that
habitual coffee consumption is associated with a substantially lower risk of Type 2
diabetes (van Dam and Hu, 2005). This association does not differ by sex, obesity, or
29 region (van Dam and Hu, 2005). Many case control studies in Asia, Northern Europe,
Southern Europe, and North America have shown consistent inverse association between
coffee consumption and the risk of colorectal cancer, although the evidence from cohort studies is inconclusive (Giovannucci, 1998). A meta analysis that combined the results of
12 case control studies also found that frequent coffee consumers had a 28 % lower risk of colorectal cancer than infrequent coffee consumers (Giovannucci, 1998). Recently, a meta analysis including 4 cohort and 5 case control studies found that an increase in
consumption of 2 cups of coffee per day was associated with a 43 % reduction in the risk of liver cancer (Larsson and Wolk, 2007). Coffee drinking has also been shown to have positive effect on neurodegenerative diseases, such as Alzheimer’s disease (Barranco
Quintana et al. , 2007). The available data suggest that this effect is due to caffeine intake
(Cunha, 2008; Arendash et al. , 2009). There is no data that has alluded to the presence of
coffee phenolics and MRPs contributing to these benefits.
In general, currently available evidence suggests that moderate amount of coffee
consumption has positive health benefits for most people. Caffeine is associated with
various aspects of mental health and brain function due to the effects on the central
nervous system. The presence of antioxidants such as CGA and MRPs may also be
important contributors for some of the health related effects attributed to coffee beverage
consumption.
30 Table 1.4 Summary of potential health benefits of coffee consumption from epidemiological studies
Health concerns Coffee consumption Relative risk Source Level of intake 1 (RR) (No. of studies) Type 2 diabetes Low * 1.00 Van Dam and Hu, 2005 Third highest * 0.94 (9 national cohort studies) Second highest * 0.72 Highest * 0.65 Colorectal cancer Low # 1.00 Giovannucci, 1998 High # 0.72 (12 case control studies) Liver cancer Per 2 cups/day 0.57 Larsson and Wolk, 2007 increment $ (4 cohort and 5 case control studies) Alzheimer’s > 0 cups/day Φ 0.73 Quintana et al ., 2007 disease (2 cohort and 2 case control studies) 1 * The low level of consumption (reference) denotes 0 cups or 2 or less cups per day; the third highest level denotes 1 to 3 cups per day, or 3 or more cups per day, or 4 to 5 cups per day; the second highest level denotes 4 to 5 cups per day, or 5 to 6 cups per day; and the highest level denotes 6 or more, or 7 or more cups per day. # The low level (reference) denotes less than 1 cups per day; and the high level denotes 4 or more cups per day. $ The estimated RR is for an increment of 2 cups of coffee per day. Φ The RR is for coffee consumers (> 0 cups per day) versus non consumers.
31 1.3 RESEARCH HYPOTHESES AND OBJECTIVES
General thesis hypothesis
MRPs exhibit different antioxidant activities that can be attributed to differences in
molecular weight and chemical character. Coffee MRPs will modulate the antioxidant
status in Caco 2 cells through the regulation of enzymatic antioxidants and genes that are
involved in oxidative stress and/or the antioxidant defense system.
General thesis objective
To characterize and compare the chemical properties of non roasted coffee, roasted
coffee, model MRPs, and related ultrafiltration fractions. To assess the associated
chemical antioxidant activities and biological effects of these products in Caco 2 cell
culture.
Experiment I. Chemical characteristics and antioxidant properties of coffee extracts
Hypothesis
1. The low molecular weight fractions (MW<1KDa) recovered from both light
roasted (LR) and dark roasted (DR) coffee beverages have greater in vitro
antioxidant potential in comparison with high molecular weight fractions
(MW>1KDa) derived from the same roasting conditions.
2. The low molecular weight components (MW<1KDa) in coffee bind non
covalently to the high molecular weight components (MW>1KDa) and thus
contribute to the in vitro antioxidant activity of the high molecular weight coffee
fractions.
32 3. Increasing the degree of roasting in coffee beverages will induce an increase in
specific antioxidant enzyme activities in cultured human Caco 2 cells.
Objective
1. To determine the molecular weight distribution of LR and DR coffee beverages
using water and sodium chloride ultrafiltration systems.
2. To qualitatively define the chemical characteristics of coffee extracts that include
color development and quality, browning intensity, UV spectra, and fluorescent
spectra.
3. To evaluate the in vitro antioxidant activities of coffee extracts, and related
ultrafiltration fractions using ORAC, ABTS and reducing power assays.
4. To investigate the affinity of coffee extracts to modify cellular chemopreventive
antioxidant enzymes (e.g. SOD, CAT, GR, GPX) activities and GSH content.
Experiment II. Coffee constituents and modulation of oxidative status in Caco-2
cells
Hypothesis
1. MRPs are the major constituents in roasted coffee brew that contribute to the
antioxidant activity of coffee.
2. Low molecular weight coffee MRPs (MW<1KDa) have greater antioxidant
activity in comparison to high molecular weight coffee MRPs (MW>1KDa).
3. Coffee MRPs vary in the affinity to increase specific antioxidant enzyme
activities in cultured human Caco 2 cells, and low molecular weight MRPs
33 (MW< 1KDa) have greater impact than high molecular weight MRPs
(MW>1KDa).
4. Coffee can modulate the oxidative status in Caco 2 cells through the regulation of
genes involved in oxidative stress and/or antioxidant defense system.
Objective
1. To determine the molecular weight distribution of extracts derived from green
coffee beans, roasted coffee beans and model MRPs using water ultrafiltration.
2. To test the chemical characteristics of coffee and coffee model MRPs, including
color development, browning intensity, UV spectra, fluorescent spectra and the
presence of α dicarbonyl compounds.
3. To evaluate the in vitro antioxidant activities of green and roasted coffee
beverages, coffee model MRPs and related ultrafiltration fractions using ORAC,
ABTS and reducing power assays.
4. To investigate the cellular in vitro antioxidant activities of coffee beverages,
coffee model MRPs and related ultrafiltration fractions by examining potential
effects on SOD, CAT, GR, GPX activity and GSH content, and the protection
against reactive oxygen species induced oxidative stress in Caco 2 cells.
5. To investigate the cellular reaction of coffee MRPs, particularly the influence on
the gene regulation of specific antioxidant enzymes and other genes involved in
oxidative stress and/or antioxidant defense system using Real time PCR array.
34
CHAPTER II
CHEMICAL CHARACTERISTERCS AND ANTIOXIDANT PROPERTIES OF
COFFEE
35 2.1 Introduction
During the process of roasting coffee, green coffee beans are heated to 200 250 ºC, for
0.75 25 min, depending on the requirement for final roasting (e.g. light, medium, or dark).
Complicated physical and chemical changes take place in the roasted coffee beans, which include thermal degradation of natural phenolic antioxidants and generation of brown, flavored compounds, called Maillard reaction products (MRPs). In lieu of the antioxidant properties that exist for both phenolics and MRPs, the roasting process of coffee will result in a different final level of antioxidant activity. Previous studies have reported that the antioxidant activity of coffee is in fact mostly dependent on the roasting conditions, much more so than brewing methods and the sources of coffee beans (Sacchetti et al. ,
2009).
The effect of roasting on the antioxidant activity of coffee brew has been thoroughly investigated by many researchers but notwithstanding this, the results have been inconsistent. For example, some studies reported an increase in antioxidant activity of coffee brew with roasting (Borrelli et al. , 2002; Sanchez Gonzalez et al. , 2005), while others found that the antioxidant activity decreased with roasting (Richelle et al. , 2001;
Borrelli et al. , 2002). Some workers concluded that medium roasted coffee has the highest antioxidant activity (del Castillo et al. , 2002; Caemmerer and Kroh, 2006), while another study found that non roasted green coffee beans possess higher antioxidant activity than the corresponding roasted samples (Daglia et al. , 2000). To complicated matters further, both green and roasted coffee beans contain a complex mixture of unknown number of chemicals, some of which may exhibit high antioxidant activity in one antioxidant test, while others exhibit a high antioxidant activity in a different test. In
36 general, MRPs derived from coffee have been demonstrated to exhibit primary
antioxidant activity towards metal prooxidant sequestering and direct free radical
scavenging activities (Wijewickreme and Kitts, 1998b; Delgado Andrade et al. , 2005;
Takenaka et al. , 2005).
The development of color due to the generation of MRPs is an important result of the
roasting of coffee beans. The HunterLab color parameters (L, a, b, ∆E) has been used to
measure the color development of yellow to brown pigments from different stages of the
Maillard reaction, and the brown intensity is also widely used to monitor the development
of MRPs (MacDougall and Granov, 1998; Morales and van Boekel, 1998; Leong and
Wedzicha, 2000; Jing and Kitts, 2004a). Few studies have been carried out on
characterizing the development of fluorescent MRPs in coffee during roasting.
The aim of the present study was to characterize the chemical properties of two coffee brews that had undergone different roasting processes. This work attempted to correlate
the differences in browning of coffee beans with antioxidant capacity using three
different chemical based assays. Emphasis was on the distribution of MRP components based on molecular weight and the contribution towards the relative antioxidant activity
of the coffee brew. Finally, an extension of these studies was performed using a cell based procedure to determine if the chemical antioxidant activity measurements indeed corresponded to biological effects that could be attributed to changes in the enzymatic antioxidant defense system of human colon adenocarcinoma Caco 2 cells. Caco 2 cells were used in this thesis for evaluating the bioactivity of coffee constituents and the effects on gastrointestinal cells (Popovich and Kitts, 2004a, b).
37 2.2 Materials and methods
2.2.1 Coffee preparation
Roasted coffee beans ( Coffea Arabica ; roasted at designated “light”: 185 °C 15 min and
“dark”: 210 °C 15 min; reference: non official company disclosure), were purchased from a local store and ground to powder (fine grind) in a standard coffee grinder. A sample of coffee powder (55 mg) was extracted with 1.1 L hot water using No.4 cellulose type coffee filters (Melitta, Canada). The fresh coffee extract obtained was rapidly cooled in an ice bath and centrifuged at 750 g for 45 min. Coffee brew supernatants (100 ml) were freeze dried and the rest (1 L) was extracted with petroleum ether (3 × 300 ml) to remove the crude lipids. The defatted coffee extract was again freeze dried and the yields of both crude and defatted extracts were determined gravimetrically. The recovered organic layer was concentrated to dryness using a rotary evaporator (Bϋchi Rotavapor R 114, Bϋchi Labortechnik AG, Flawil, Switzerland) under vacuum at 40 ºC and the yield was recorded. Samples were stored at 4 °C until analysis was conducted.
2.2.2 Ultrafiltration
Freeze dried defatted coffee extracts were dissolved in water, or in 2 M NaCl and fractionated by multiple step ultrafiltration (Millipore, USA). The molecular weight cut off for each fraction was: 10KDa (YM 10), 1KDa (YM1), and 0.5KDa (YC 500), respectively. In a different experiment, NaCl was used to release the low molecular weight compounds ionically bound to the high molecular weight component.
Ultrafiltration separation was performed on the samples under a nitrogen pressure of 40
38 psi and individual fractions were collected and freeze dried. The residues for molecular
weight fractions, Fraction I (MW>10KDa), Fraction II (1KDa III (0.5KDa °C until analysis. 2.2.3 Physical chemical analyses 2.2.3.1 Measurement of color Color analyses on the ground coffee samples, crude and defatted coffee extracts were performed using a HunterLab Labscan 600 spectrocolorimeter (Hunter Associates Lboratory Inc., Reston, Virginia). The instrument was calibrated with black and white tiles. Color was expressed in L (L = 0 yields black and L = 100 indicates diffuse white), a (negative values indicate green and positive values indicate red), b (negative values indicates blue and positive values indicate yellow) Hunter scale parameters. The colorimetric difference E was obtained through the equation: E = [(L)2 + (a)2 + (b)2]0.5 . Five measurements were carried out on each sample. 2.2.3.2 Measurement of browning and UV vis spectra Coffee brew samples were dissolved in distilled water at 0.5 mg/ml and 200 l of each sample was placed into a 96 well plate for the test. The UV vis absorbance over the range of 250 700 nm was recorded with a 5 nm interval. Indices of browning of the coffee extracts and related fractions were determined using an absorbance maximum set at 420 nm (Multiskan Spectrum, ThermoLabsystem, Helsinki, Finland). A blank, containing only distilled water was used to correct absorption readings. 39 2.2.3.3 Measurement of fluorescence Coffee brew samples were dissolved in 3 ml of Milli Q water (0.25 mg/ml), to prevent quenching effects. The solution was then measured at an excitation wavelength of 400 nm and emission wavelength range from 350 to 550 nm using a Shimadzu RF 5301 spectrofluorophotometer (Kyoto, Japan). An average of three readings was recorded. 2.2.4 Chemical based antioxidant assays 2.2.4.1 Trolox equivalent antioxidant capacity (TEAC) assay ABTS [2, 2´ Azino bis (3 ethylbenzothiazoneline 6 sulfonic acid)] radical cation (ABTS•+) stock solution was prepared by mixing 5 ml of 7 mM ABTS (Sigma, St. Louis, MO, USA) with 88 l of 140 mM potassium persulfate. This mixture was allowed to remain in the dark, at room temperature for 12 24 h until the reaction was complete and the absorbance was stable. Fresh ABTS•+ working solution was prepared for each assay by mixing 600 l ABTS•+ stock solution with 40 ml distilled water to obtain an absorbance of at least 0.4 at 734 nm. The ABTS radical scavenging effect of coffee extracts at different concentrations (0 1.0 mg/ml) was calculated using the following equation: % inhibition= (1 absorbance sample /absorbance control ) ×100 The standard curve was linear between 0 25 mM Trolox (Sigma Aldrich, Oakville, ON, Canada). Trolox equivalent antioxidant capacity (TEAC) =slope sample /slope control . Results were expressed in mmol Trolox equivalent (TE)/g sample. 40 2.2.4.2 Oxygen radical absorbance capacity (ORAC) assay ORAC assay measures the ability of antioxidant components in test materials that inhibit the decline in fluorescence induced by a peroxyl radical generator, AAPH (2, 2 azobis (2 amidinopropane) dihydrochloride) (Wako Chemcal Inc., Richmond, VA, USA). The following reactants were added to each well in 96 well black walled plates: 100 l sample (final concentrations of 0 1.0 g/ml) in 75 mM phosphate buffer (pH 7.0) or Trolox standard (final concentrations of 0 6.0 M) and 60ml fluorescein (Sigma, St. Louis, MO, USA) (final concentration 60 nM). Each plate was incubated at 37 ºC for 15min; then 60 l AAPH (final concentrations 12 mM) were added and fluorescence readings (Excitement wavelength = 485 nm, Emssion wavelength = 527 nm) were continuously taken (0 60 min) using a fluorescence microplate reader (Huoroskan Ascent FL, Labsystems). Data transformation and interception were performed according to the method of Davalos et al. (2004) and ORAC values were expressed as mmol TE/g sample. 2.2.4.3 Reducing power (Gu’s RP assay) Reducing power of the sample was tested using the method of Gu et al. (2009a) with some modification. Aliquots (e.g. 300 l) of samples over a concentration range of 0 2.0 mg/ml and standard chlorogenic acid solutions (CGA; Sigma, St. Louis, MO, USA) (0 1.0 mg/ml) were mixed with 300 l phosphate buffer (0.2 M, pH 6.6) and 300 l potassium ferricyanide (BDH, Product Code. B10204). The mixture was incubated at 50ºC for 20min. The reaction was terminated by adding trichloroethanoic acid (TCA; Fisher, Nepean, ON, USA) solution (10 % w/v) and centrifuged at 3,000 rpm for 10 min. 60 l of the supernatant was mixed with 60 l distilled water and 12 l ferric chloride (0.1% FeCl 3) in a 96 well plate, incubated for 8 min in the dark, and the absorbance was 41 measured at 700 nm. Reducing power was expressed as g chlorogenic acid (CGA)/g sample. 2.2.5 Cell based assays 2.2.5.1 Cell culture Caco 2 cells were obtained from ATCC (Manassas, VA) and maintained in Minimum Essential Medium (MEM, Sigma, St. Louis, MO, USA), which were supplemented with 10 % fetal bovine serum (FBS, Gibco, Grand Island, NY. USA), penicillin (100 U) and streptomycin (100 g/ml) (Gibco, Grand Island, NY. USA). Cells (passage 24 40) were cultured in an incubator (37ºC) under an atmosphere of 5 % CO 2 with 90 % humidity. Culture media were changed every 48 h. For the measurement of cell viability, cells were seeded in 96 well plates at a density of 5 × 10 5 cells/ml 24 h before coffee treatments. For enzyme activities and glutathione status, cells were seeded in a cell culture Petri dish at a density of approximately 1.0 × 10 5 cells/ml. Cells were allowed to reach 80 % confluence, which took about 5 days in culture at the time of treatment. At sub confluence, cells were in homogeneously undifferentiated (Vachon and Beaulieu, 1992). 2.2.5.2 Cell counting Culture medium was removed and 0.25 % Trypsin EDTA (Gibco, Grand Island, NY. USA) was added to cells. Cells were incubated at 37 ºC until detachment from the plates occurred. Cells were manually dispersed to attain a single cell suspension. The trypsin was neutralized by adding fresh culture media to cells, which was followed by cell counting using a haemocytometer. Viable cells were assessed by trypan blue exclusion dye (Sigma, UK.). 42 2.2.5.3 Coffee treatment of Caco 2 cells Caco 2 Cells were exposed to filter sterilized defatted coffee extracts (0.1 mg/ml in culture medium) at different incubation times. The treated cells were rinsed with ice cold PBS and then scraped into a 1.5 ml tube. The cell samples were put through a freeze (liquid nitrogen, 2 min) thaw (37 ºC, 5 min) cycle, 3 times, to release cytosol constituents and then centrifuged at 4ºC at 15,000 g for 10 min. The supernatant was decanted into a new tube, adjusted to a final volume of 700 l, and the cell pellet was discarded. The supernatants were kept on ice prior to protein content, enzyme activity and glutathione status measurements. 2.2.5.4 MTT cell viability assay Following incubation of cells with coffee extracts at different concentrations for different incubation periods, Caco 2 cells were rinsed with phosphate buffer saline (PBS, pH 7.2). A medium containing MTT (0.5 mg/ml; [3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyl tetrazolium bromide], Sigma, St. Louis, MO, USA) was added to the cell culture. Cells were incubated in the dark for 4 h with the MTT medium. To solubilize the formazan crystal, SDS (10 %) in hydrochloric acid (HCl) (0.1 N) was added and the plates were incubated overnight. Optical density readings were taken at 570 nm in a microplate reader (ThermoLabsystems Multiscan Spectrum, Thermolabsystem, Chantilly, VA). Absorbance values measured at 570 nm were corrected for background absorbance using well that containing only MTT medium. Cell MTT response (% control) was calculated from the equation: % control = absorbance treatment /absorbance control × 100% 43 2.2.5.5 Protein content Protein content of the Caco 2 cell supernatants was measured according to the method of Bradford using bovine serum albumin (BSA) as the standard protein (Bradford, 1976). Briefly, 5 l of standard, sample or blank was mixed with 250 l Bradford dye reagent. After 45 min incubation, the absorbance was read in a 96 well plate in triplicate using a microplate reader at 595 nm. The standard curve was prepared in a range between 0.1 and 1.4 mg/ml BSA in PBS. 2.2.5.6 Glutathione status Glutathione (GSH) status was measured by determining 5 thio 2 nitrobenzoate (TNB) generation, resulting from the reaction of 5, 5’ dithiobis (2 nitrobenzoic acid) with GSH (Anderson, 1985). Coffee treated and untreated cell supernatants were first deproteinized with 5% 5 sulfosalicylic acid (SSA) solution (2:1 v/v), centrifuged to remove the precipitated protein and then assayed for GSH using the enzymatic procedure. In a 96 well plate, 10 l of supernatant was added to 240 l of a freshly prepared reaction mixture, containing 30 l of 6 mM 5, 5’ dithiobis (2 nitrobenzoic acid) (DTNB) (Sigma, St. Louis, MO, USA), and 210 l working solution [(0.248 mg/ml β nicotinamide adenine dinucleotide phosphate (NADPH; Sigma, St. Louis, MO, USA) in a sodium phosphate buffer (143 mM, pH 7.5)] and incubated at 37 ºC for 20 min. GSSH reductase (0.5 U; Sigma, St. Louis, MO, USA), was added to initiate the assay. The reaction mixture, without sample, was used as a blank. TNB formation rate of both samples and GSH standards (Sigma, St. Louis, MO, USA), were monitored using a microplate reader at 412 nm at 1 min intervals for 5 min. Cellular GSH concentrations (nmol/mg protein) were determined from a standard curve of nanomoles of GSH equivalents versus rate of 44 change in activity (e.g. change in absorbance/min). The calculations are shown below. Means and ranges for SD were obtained from two or more independent experiments. Each experiment was performed in duplicate. nmoles GSH per mg of sample = A 412 /min (sample) × df A 412 /min (1 nmole) × pc Where A 412 /min (sample) = slope generated by sample A 412 /min (1 nmole) = slope calculated from standard curve for 1 nmole of GSH df = dilution factor of original sample pc= protein content of sample in the reaction in mg 2.2.5.7 AntioxidanteEnzyme assays Catalase (CAT) activity was determined using the UV spectrophotometric assay reported by (Claiborne, 1985) with some modifications. A volume of 4.5 l of 50 % ethanol was added to 225 l of cell supernatant and incubated on ice for 30 min. This was followed by the addition of 10 % Triton X 100 to a final concentration of 1.0 %, and mixed thoroughly. To initiate the reaction, a working solution (275 l) of 20 mM hydrogen peroxide (Fisher, Nepean, ON) in 50 mM phosphate buffer (pH 7.0) was added to individual wells on a 96 well UV plate, followed by 125 l of the 10 times diluted cell supernatant. Absorbance readings were monitored at 240 nm at 25 °C for 2 min with a reference containing only the working solution. Catalase activity in samples was expressed as micromoles of hydrogen peroxide consumed per minute per milligram of protein. Superoxide dismutase (SOD) activity was measured using a SOD Assay Kit WST obtained from Dojindo (Gaithersburg, MD, USA). Generally, 100 l of 45 chloroform/ethanol (3/5, v/v) was added to 250 l of cell supernatant with vigorous mixing. The mixture was centrifuged at 1400 g at 4 ºC for 10 min. The supernatant was transferred into a new tube and the pellet was discarded. An aliquot of supernatant (e.g. 20 l) from different dilutions was incubated at 37 °C for 20 min with a reaction mixture containing xanthine, xanthine oxidase, and (2 (4 Iodophenyl) 3 (4 nitrophenyl) 5 (2,4 disulfophenyl) 2H tetrazolium, monosodium salt) (WST 1). Absorbance readings at 450 nm were recorded using a microplate reader. SOD derived from bovine erythrocytes (Sigma, St. Louis, MO, USA) was used to make the standard curve. SOD activities of the samples were calculated based on the SOD standard. The definition of SOD Unit, according to Sigma product information, is that one unit inhibits the rate of reduction of cytochrome c by 50 % in a coupled system, using xanthine and xanthine oxidase. The glutathione peroxidase (GPX) activity assay was performed by adding cell supernatant samples into a working solution that included 1 mM glutathione, 2 mM sodium azide (Fisher, Nepean, ON), 1 U/mL glutathione reductase, 0.1 mM β nicotinamide adenine dinucleotide phosphate (NADPH). The reaction medium was incubated for 5 min at 37 ºC before initiating the reaction with the addition of 10 l of 7.5 M hydrogen peroxide. The disappearance of NADPH absorbance at 340 nm was monitored for 3 min (Paglia and Valentine, 1967). A non enzymatic reaction rate (blank) was determined by substituting water for cell supernatant and recording the decrease in NADPH absorbance. One unit of GPX activity was defined to be equivalent to the oxidation of 1 nanomole NADPH per minute per milligram protein. The glutathione reductase (GR) activity assay was determined using a Glutathione Reductase assay kit obtained from Sigma (St. Louis, MO, USA). This assay is based on 46 the reduction of glutathione (GSSG) by NADPH in the presence of glutathione reductase. In addition, DTNB reacts spontaneously with the reduced glutathione (GSH) and generates 5 thio (2 nitrobenzoic acid) (TNB), which can be measured by the increase in absorbance at 412 nm using an extinction coefficient (ε mM ) of 14.15 for TNB. One unit causes the reduction of 1.0 mole of DTNB to TNB at 37 °C. 2.2.6 Statistical analysis Each experiment was performed in triplicate (e.g. three wells or three cell culture plates) and repeated three times in separated experiments. Collected data were expressed as mean + SD. Data were analyzed by One way Analysis of Variance (ANOVA), followed by Tukey’s pairwise comparisons. Comparison of multiple treatments with control was done by one way ANOVA, followed by Dunnett’ test. Significant differences between two samples were analyzed with Student t test. In some cases, data were analyzed with two way ANOVA followed by Bonferroni post tests. The level of confidence required for significance was selected at p<0.05. 47 2. 3 Results 2. 3.1 Yields and recovery of coffee brews and ultrafiltration fractions Freshly brewed coffee processed at specific temperature and time conditions required to produce a light roast (LR) and dark roast (DR), respectively, resulted in a final yield of crude coffee extracts that was 13.48 g/L and 14.68 g/L, respectively (Table 2.1). The weight of total crude lipids recovered from coffee brews defatted by triple extraction with petroleum ether was not significantly different between LR and DR coffee extracts. Similar yields of hydrophilic constituents were also obtained in LR and DR coffee extracts. Water ultrafiltration was used to recover four distinct fractions of the hydrophilic coffee extracts with molecular weights that ranged from >10KDa (Fraction I); 1 10KDa (Fraction II); 0.5 1KDa (Fraction III) and <0.5KDa (Fraction IV). The recovery of freeze dried fractions is presented in Table 2.2. A relatively greater proportion (p<0.05) of the coffee brew constituents was found in the low molecular weight fractions (e.g. Fractions III and IV). Total recovery of all coffee brew constituents in the four molecular weight fractions was 97 % and 96 %, for LR and DR, respectively. In order to remove non covalent bound low molecular weight compounds from the melanoidin skeleton, the defatted coffee extracts were also treated with 2 M NaCl, followed by similar multistep ultrafiltration. The recovery of all coffee constituents present in the salt Fraction I S to IV S (I S: S = salt) are presented in Table 2.2. Fraction IVS was not able to be recovered due to the excess salt content. A correction, therefore that consisted of summing the recovery of the three fractions and expressing this as 100 percent recovery was used to calculate and estimate Fraction IV S recovery. The recovery from the salted Fraction IVS was found to 48 be numerically greater (p<0.05) than that recovered in the non salted Fraction IV using water ultrafitration. Thus, a greater recovery of low molecular weight compounds (MW<0.5KDa) was made when coffee brew extracts were treated with salt to liberate non covalently bound compounds. Table 2.1 Recovery yields of coffee extracts 1 Coffee extracts Yield 2 LR DR Crude extract 13.48+0.77 b 14.68+0.28 b Defatted extract 12.96+0.38 b 13.87+0.12 b Total crude lipid 0.37+0.16 a 0.67+0.09 a 1 Values are expressed as mean + SD, n=3. ab represent means in columns that are significantly different. Statistical analyses were done by two way ANOVA with Bonferroni post tests; level of confidence set as 0.05. 2 Yields of crude, defatted extracts and total crude lipids are expressed as g/L of coffee brew. Table 2.2 Recovery of coffee fractions by water and salt ultrafiltrations 1 Fractions (MW) 2 LR DR water salt water salt I (>10KDa) 17.7+0.6 a 15.5+5.9 b 19.4+1.5 ab 18.0+4.6 a II (1 10KDa) 13.8+2.2 a 8.0+2.5 a 15.6+2.7 a 12.5+1.9 a III (0.5 1KDa) 30.7+0.9 b 28.6+4.5 c 21.5+2.2 b 18.8+2.5 a IV (<0.5 KDa) 3 34.9+5.4 bx 48.0+1.1 dy 39.8+2.3 cx 50.7+3.7 by 1 The recovery of fractions are expressed as % of defatted extracts dry weight (mean + SD, n=3). ab represent means in columns that are significantly different; xy represent different means in rows between water and salt ultrafiltration. Statistical anaylyses were done by two way ANOVA with Bonferroni post tests; level of confidence set as 0.05. 2 Fractions were derived from defatted extracts, based on molecular weight (MW). 3 The recovery of Fraction IV derived from salt ultrafiltration system was calculated using this equation: 100 – R Fraction I – R Fraction II – R Fraction III ; R = recovery. 49 2.3.2 Chemical characteristics of coffee 2.3.2.1 Colorimetric measurements (L a b analysis) Quantitative colorimetric measurements of LR and DR ground coffee powder, and freeze dried crude and defatted extracts and related ultrafiltration fractions were obtained to assess differences in color. The measure of total color difference ( E), reveals all colorimetric parameters such as chroma and lightness (L). Significantly higher L and E values were observed in the ground powder and freeze dried crude extracts derived from LR coffee beans compared to those from DR beans (Table 2.3). However, no significant differences in L and E values were observed between LR and DR coffee defatted extracts. Colorimetric parameters of water ultrafiltration fractions derived from defatted coffee extracts are present in Table 2.4. It was found that a pattern of relative increase in both L and E corresponded to a decreased molecular weight in the four fractions. Table 2.3 Color parameters (L, E) of coffee ground powder and extracts 1 Coffee extracts L E LR DR LR DR Ground power 16.2+1.0 ay 11.3+0.5 ax 21.3+1.0 ay 13.3+0.6 ax Crude extract 35.5+2.3 cy 30.5+1.2 bx 43.0+3.0 cy 37.8+1.6 bx Defatted extract 31.7+0.7 b 29.5+2.2 b 38.7+0.7 b 35.8+2.7 b 1 Values are expressed as mean + SD, n=3. abc represent means in columns that are significantly different; xy represent significantly different means in rows between LR and DR. Statistical analyses were done by two way ANOVA with Bonferroni post tests; level of confidence set as 0.05. Color difference is expressed as E = (L 2 + a 2 +b 2)1/2 , where “L” represents lightness, “a” represents red/green, and “b” represents yellow/blue. 2.3.2.2 Browning of coffee extracts (UV analysis) The extent of browning in LR and DR coffee ultrafiltration fractions is shown in Table 2.4. There was no significant difference observed for UV absorbance between defatted 50 LR and DR coffee extracts. The degree of browning found in Fractions I and II was significantly higher (p<0.05) than that of fractions III and IV. No significant difference in browning was found between Fractions I and II, and between low molecular weight Fractions III and IV. Table 2.4 Color parameters (L, E) and browning of fractionated coffee extracts 1 Fractions(MW) 2 L E 3 Browning 4 LR DR LR DR LR DR I (>10KDa) 29.5+3.1 a 23.0+0.8 a 34.7+3.5 ay 27.1+0.9 ax 0.54+0.07 b 0.54+0.07 b II (1 10KDa) 35.4+1.4 ab 32.7+2.7 b 43.1+1.8 b 39.3+2.5 b 0.50+0.09 b 0.39+0.08 b III (0.5 1KDa) 37.3+2.9 bc 32.9+5.5 b 52.3+3.9 cy 40.2+3.9 bx 0.13+0.02 a 0.14+0.00 a IV (<0.5 KDa) 42.5+3.7 c 46.0+5.2 c 56.2+3.0 c 60.7+3.2 c 0.07+0.00 a 0.07+0.00 a 1 LR and DR represent light roasted and dark roasted coffee. Values represent mean + SD, n = 3. abcd The means with different superscript letters in each column are significantly different; xy represent significantly different means in rows between LR and DR (p<0.05, two way ANOVA with Bonferroni post tests). 2 Fractions are derived from defatted extracts, based on molecular weight (MW). 3 Color difference is expressed as E = (L 2 + a 2 +b 2)1/2 , where “L” represents lightness, “a” represents red/green, and “b” represents yellow/blue. 4 Browning intensities are absorbance readings at 420 nm. Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 2.3.2.3 Fluoresence and UV vis spectra The defatted extracts and fractions of different molecular weight components derived from coffee brews produced similar spectral fluorescence patterns with a sharp peak occurring at 400 nm and another broad peak also observed at higher emission wavelengths (Figure 2.1). The intensity of the first peak at 400 nm in Fraction I was greater than that found for other fractions. In general, the fluorescence intensity for different coffee fractions did not correspond to the trend found between increased molecular weight and the degree of browning and tri stimulus colorimetry for Fractions I IV. 51 The UV absorbance spectra measured over a 250 700 nm range was obtained for both LR and DR coffee defatted extracts and ultrafiltration fractions (Figure 2.2). There was no peak absorption associated with Fraction I for both LR and DR coffee extracts. Fraction II had a characteristically higher absorbance intensity but a similar pattern existed between LR and DR coffee extracts. In contrast, the absorbance spectra for Fraction III contained distinct absorbance maxima at 285 nm and 320 nm for LR coffee; while the DR coffee had an absorbance maximum at 280 nm (Figure 2.2D). A clear difference in the intensity of absorbance spectra were found in Fraction IV between LR and DR. Maximal absorbance values recorded at 275 nm were greater for LR than DR (p<0.05). 52 Figure 2.1 Fluorescence emission spectra (350 550 nm) of light roasted (LR ) and dark roasted (DR ) coffee extracts and fractions. Defatted coffee extracts (A), Fraction I (B), Fraction II (C), Fraction III (D) and Fraction IV(E), measured with the excitation wavelength set at 400 nm. Samples were diluted to 0.25 mg/ml with mili Q water prior to spectrum measurement. 53 Figure 2.2 Comparison of the UV visible spectra of light roasted (LR ) and dark roasted (DR ) coffee extracts and fractions. Defatted coffee extracts (A), Fraction I (B), Fraction II (C), Fraction III (D) and Fraction IV(E), measured from 250 700 nm absorbance wavelength. Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 54 2.3.3 Antioxidant activity of coffee extracts in chemical systems Tables 2.4 2.6 present the results of antioxidant activity measurements on coffee samples using three selected methodologies. ORAC and TEAC data are expressed as mmol equivalents of Trolox (TE)/ g of sample, a commonly used unit for antioxidant assays. Chlorogenic acid (CGA) equivalent was used for the RP assay, due to the presence of this phenolic in coffee and relative important contribution to the overall antioxidant capacity. The antioxidant activity of LR and DR coffee was compared to see the influence of roasting conditions. Generally, LR coffee had greater (p<0.05) antioxidant activity compared to DR, based on the results from the three assays (Table 2.4). There was no significant difference in antioxidant activity between crude and defatted coffee brews, as measured by the three assays (Table 2.4), thus indicating that the hydrophilic components in coffee brew were mainly responsible for the observed antioxidant activity. However, significant differences in the antioxidant activity were found between different molecular weight fractions. For the ORAC assay, Fractions III and IV had higher TE values in comparison to the corresponding Fractions I and II (p<0.05), thus indicating a higher peroxyl radical scavenging ability associated with low molecular weight constituents (MW<1KDa). For the TEAC assay, Fraction I exhibited the lowest ABTS radical scavenging capacity (p<0.05), and Fractions II and III had the highest TEAC values (p<0.05). Fraction I had the lowest reducing activity. Fraction III had the highest antioxidant capacity based on all antioxidant measurements. In order to evaluate the efficiency of ultrafiltration at maintaining the antioxidant activity of different coffee components present in all fractions, Fractions I, II, III and IV were combined in proportion to form a recombined fraction. No significant difference in the antioxidant 55 activity between the recombined fraction and the non fractionated defatted extract was obtained in all three assays (Table 2.5). This result indicated that the antioxidant activity of coffee components was maintained after ultrafiltration. Samples that were treated with 2 M NaCl to release low molecular weight compounds ionically attached to the melanoidin skeleton were also assayed for antioxidant activities. In the DR coffee extracts, TEAC and RP, but not ORAC, increased significantly (p<0.05) in Fraction I S after salt treatment. In contrast, Fraction I S recovered from the LR coffee extract showed increased ORAC and TEAC values (p<0.05) compared to the non salt treated fraction I. For Fraction II S, both TEAC and RP values were significantly higher (p<0.05) in LR and DR extracts compared to non salt treated samples. The ORAC value taken for Fraction III S of DR coffee extract was less than non salt treated Fraction III, while the TEAC and RP values were significantly higher (p<0.05). For LR Fraction III S, all the three values were significantly lower compared to non salt treated samples (p<0.05). 56 Table 2.4 Antioxidant activities of coffee extracts and fractions 1 Coffee extract Assays 2 ORAC TEAC RP LR DR LR DR LR DR Crude 1.61+0.08 y 1.32+0.09 x 0.54+0.02 y 0.47+0.04 x 0.21+0.01 y 0.13+0.02 x Defatted 1.61+0.03 y 1.37+0.09 x 0.60+0.01 y 0.47+0.02 x 0.21+0.00 y 0.15+0.01 x Fractions (MW) I (>10KDa) 0.51+0.04 a 0.64+0.03 a 0.38+0.02 a 0.37+0.01 a 0.14+0.01 ay 0.11+0.00 ax II (1 10KDa) 1.25+0.06 b 1.31+0.06 b 0.62+0.04 cy 0.56+0.03 cx 0.17+0.01 b 0.15+0.01 b III (0.5 1KDa) 2.02+0.12 cy 1.82+0.09 cx 0.59+0.01 c 0.55+0.02 c 0.25+0.01 dy 0.15+0.01 bx IV (<0.5 KDa) 1.89+0.10 c 1.88+0.11 c 0.51+0.02 b 0.46+0.03 b 0.20+0.01 cy 0.14+0.01 bx 1 DR and LR represent dark roasted and light roasted coffee. Value represents mean + SD (n=3), significant differences were analyzed with two way ANOVA with Bonferroni post tests. abc represent means in columns that are significantly different; xy represent significantly different means in rows between light roasted (LR) and dark roasted (DR). 2 ORAC represents oxygen radical absorption capacity; TEAC represents Trolox equivalent antioxidant capacity; RP represents reducing power. ORAC and TEAC values are expressed as mmol Trolox equivalents/g freeze dried coffee samples; RP values are expressed as mg CGA/g freeze dried coffee samples. 57 Table 2.5 Antioxidant activity of defatted non fractionated coffee extracts and recombined extracts 1 Coffee extract Assays 2 ORAC TEAC RP LR DR LR DR LR DR Non fractionated 1.61+0.03 y 1.37+0.09 x 0.60+0.01 y 0.47+0.02 x 0.21+0.00 y 0.15+0.01 x Recombined 3 1.64+0.14 y 1.39+0.10 x 0.61+0.04 y 0.46+0.03 x 0.19+0.01 y 0.14+0.01 x 1 Value represents mean + SD (n=3), significant differences were analyzed with two way ANOVA with Bonferroni post tests. xy represent significantly different means in rows between LR and DR. No significant differences between defatted and fraction I IV. 2 ORAC represents oxygen radical absorption capacity; TEAC represents Trolox equivalent antioxidant capacity; RP represents reducing power. ORAC and TEAC values are expressed as mmol Trolox equivalents/g freeze dried coffee samples; RP values are expressed as mg CGA/g freeze dried coffee samples. 3 Recombined extract represents recombined fraction I, II, III and IV. 58 Table 2.6 Antioxidant activities of coffee fractions by water and salt ultrafiltration 1 Fractions 2 Assays 3 ORAC TEAC RP LR DR LR DR LR DR I 0.51+0.04 a 0.64+0.03 0.38+0.02 a 0.37+0.01 a 0.14+0.01 y 0.11+0.00 ax S by x b b by I 0.97+0.06 0.66+0.13 0.54+0.04 0.56+0.04 0.15+0.01 x 0.17+0.01 II 1.25+0.06 a 1.31+0.06 0.62+0.04 a 0.56+0.03 a 0.17+0.01 ay 0.15+0.01 ax S by x b b bx II 1.58+0.15 1.17+0.09 0.77+0.07 0.68+0.03 0.23+0.01 by 0.21+0.02 III 2.02+0.12 by 1.82+0.09 bx 0.59+0.01 by 0.55+0.02 ax 0.25+0.01 by 0.15+0.01 ax S III 1.23+0.05 a 1.08+0.08 a 0.51+0.01 ax 0.66+0.01 by 0.13+0.00 ax 0.21+0.00 by 1 Value represents mean + SD (n=3), significant differences were analyzed with two way ANOVA with Bonferroni post tests. ab represent means in columns between fraction and fraction S that are significantly different; xy represent means in rows between light roasted (LR) and dark roasted (DR) that are significantly different. 2 Fractions were derived from defatted coffee extracts using water (I, II, III) or salt (I S, II S, III S), based on molecular weight (MW). 3 ORAC represents oxygen radical absorption capacity; TEAC represents Trolox equivalent antioxidant capacity; RP represents reducing power. ORAC and TEAC values are expressed as mmol Trolox equivalents/g freeze dried coffee samples; RP values are expressed as mg CGA/g freeze dried coffee samples. 59 2.3.4 Biological effects of coffee extracts 2.3.4.1 MTT response The potential cytotoxicity of coffee was tested using defatted coffee extracts on Caco 2 cells. The Caco 2 cell MTT response to coffee extracts (LR and DR) was found to be dependent on both the incubation time and sample concentration (Figure 2.3). Cells showed reduced (p<0.05) viability when incubated with 2.5 mg/ml coffee (LR and DR) for 3 h. This toxicity potential was much more severe when cells were exposed to coffee extracts for longer times. For example, after 72 h treatment, cells showed a reduced viability at concentrations as low as 0.25 mg/ml coffee treatment (p<0.05). There were no signs of a toxic effect when cells were treated with coffee extracts at concentrations lower than 0.1 mg/ml for all incubation time. The IC 50 for different treatment durations of LR and DR coffee extracts, derived from the concentration response MTT curves, is summarized in Table 2.7. 60 Figure 2.3 Effects of light roasted (LR) and dark roasted (DR) coffee extracts on the tetrazolium reduction rate in the MTT assay after 3 h ( ), 12 h ( ), 24 h ( ), 48 h ( ) and 72 h ( ) incubation. A: LR; B: DR. Data (% control) represent means + SD obtained for triplicate samples measured in triplicate. 61 1 Table 2.7 IC 50 of coffee extracts on Caco 2 cells using MTT assay Time IC 50 (mg/ml) LR DR 3h 5.76+0.33 d 5.29+0.32 d 12h 2.94+0.20 c 3.12+0.15 c 24h 1.58+0.09 b 1.65+0.09 b 48h 1.27+0.07 a 1.24+0.06 a 72h 0.96+0.06 a 0.99+0.05 a 1 IC 50 is determined as the concentration of coffee that was required to reduce cell viability to half that of control in the MTT assay. Values represent means + SD obtained for triplicate samples measured in triplicate. abcd represent means in columns that are significantly different. No significant difference between light roasted (LR) and dark roasted (DR). Statistical analyses were done by two way ANOVA with Bonferroni post tests; level of confidence set at 0.05. 2.3.4.2 Antioxidant enzyme activity There was no significant change in the cellular antioxidant enzyme activities of GPX, GP, and SOD (data in Appendix Table 1 3), following exposure to 0.1 mg/ml LR and DR coffee at different incubation time periods. The activity of CAT was not changed significantly in Caco 2 cells following 3 h and 24 h coffee treatment, however, it was decreased significantly (p<0.05) after 72 h of LR coffee exposure (Figure 2.4). A similar result was not obtained with cells exposed to the DR coffee extract. 62 Figure 2.4 Effect of coffee extracts on catalase (CAT) activity in Caco 2 cells. Cells were exposed to 0.1 mg/ml light roasted (LR) and dark roasted (DR) coffee for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. * indicates significant difference compared to corresponding control (p<0.05). 63 2.3.4.3 Intracellular antioxidant status As an index of the intracellular antioxidant defense, the concentration of total glutathione (GSH) was measured in Caco 2 cells treated with 0.1 mg/ml LR and DR coffee extracts for different time periods (Figure 2.5). No significant change in cellular GSH content was found after 3 h exposure to LR and DR coffee treatments, respectively. A significant decrease (p<0.05) in GSH content was observed in cells treated with LR coffee for 24 h. DR coffee treated cells showed a trend for a lower GSH content, but this effect was not significant. After 72 h exposure of cells to LR and DR coffee defatted extracts, a trend for reduced cellular GSH content was observed; however, the effect was again not significant. 64 Figure 2.5 Effect of coffee extracts on glutathione (GSH) content in Caco 2 cells. Cell were exposed to 0.1 mg/ml light roasted (LR) and dark roasted (DR) coffee for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. * indicates significant difference compared to corresponding control (p<0.05). 65 2.4 Discussion 2.4.1 Chemical characteristics of coffee Color change is an important phenomenon during the roasting process of green coffee beans. This is mostly due to the Maillard browning reaction (Massini et al. , 1990). Colorimetric measurement is a simple and widely used technique to describe the roasting degree of coffee, which is classified as light, medium and dark according to a lightness parameter L. During the roasting process of coffee beans, lightness parameter L has been found to be negatively correlated to the roasting time at a given temperature and a linear correlation between the reciprocal of lightness and roasting time has also been reported (Gokmen and Senyuva, 2006; Sacchetti et al. , 2009). In the present study, greater lightness and total color ( E) were associated with LR coffee beans that were roasted at a lower temperature compared to DR beans. In addition, this result shows that freeze dried coffee extracts, which contain mainly water soluble MRPs, have greater lightness and total color compared to ground coffee powder, which contain both water soluble and insoluble MRPs. The color differences (L and E) between LR and DR coffee extracts were not significant after delipidation, indicating some contributions of the lipophilic coffee components to the color appearance of coffee extracts. These results also indicate that the color parameters (L and E) of water soluble MRPs derived from LR coffee extract are not significantly different from DR coffee MRPs when roasted under similar roasting conditions with the only exception of temperature. The present work suggests that both the color parameters (L and E) and the degree of browning of coffee MRPs are related to complex mixtures of MRPs components and their molecular weights. High molecular weight MRPs had a greater degree of darkness and browning, while greater 66 lightness and less browning was associated with low molecular weight MRPs. The low molecular weight components of browning have been demonstrated to be generated in the early stage of the Maillard reaction (Hayashi and Namiki, 1986). Both fluorescence and UV absorbance have been used to characterize MRPs (Pongor et al. , 1984; Wijewickreme et al. , 1997). In the case of coffee, at least two groups of compounds with different chemical structures showed typical patterns in the fluorescent spectra. One group that prevailed in the high molecular weight (MW>10KDa) fraction, and to a lesser extent in the other three fractions (MW<10KDa) had a sharp peak at 400 nm wavelength. The broad peaks that occurred at higher emission wavelengths (e.g. 450 500 nm) indicated that many fluorescent constituents were present in Fractions II, III and IV derived from coffee extracts. Similar fluorescence spectra have been shown for different sugar Lys model MRPs (Jing and Kitts, 2004a). The UV absorbance spectra of the high molecular weight (MW>1KDa) fractions derived from both LR and DR coffee extracts had different patterns compared to absorption spectra of low molecular weight (MW<1KDa) fractions. High molecular weight coffee components, mostly late stage MRPs produced absorbance throughout the whole wavelength spectra (250 700 nm), but did not exhibit a clear peak absorbance pattern. This result indicates that numerous chromophores are presented in high the molecular weight coffee MRPs fractions. The finding is also supported by a previous study conducted with different sugar amino acids model MRPs (MacDougall and Granov, 1998). Low molecular weight coffee components (MW<1KDa) that had two peaks at 275 285 nm and 320 nm are similar to reports of low molecular weight MRPs that maximized at 280 290 nm (Obretenov et al. , 1986; Bailey et al. , 1996; Jing and Kitts, 2004a). Taken together, these results indicate that low molecular 67 weight coffee MRPs have distinct UV absorbance at around 280 nm, which may correlate to a similar chemical group. Results from the present study also indicate that low molecular weight coffee MRPs are likely transformed or degraded further under severe DR conditions. 2.4.2 Antioxidant activity of coffee extracts Data from the present work demonstrated that both LR and DR coffee extracts contained components that had strong overall antioxidant properties, which can be mainly attributed to the low molecular weight components (MW<1KDa). Fractions III and IV derived from both LR and DR coffee extracts, containing compounds having molecular weight below 1KDa, and these contributed about 80 % of the total peroxyl radical scavenging activity, and about 70 % of the total ABTS •+ scavenging ability. Reducing power of the defatted coffee extracts was also similar for LR (76 %) and DR (66 %). Our results agree with others reports using a linoleic acid peroxidation system, where 78 % of the overall inhibitory effect of coffee brew was found in the low molecular weight fraction (MW<1KDa) (Somoza et al. , 2003). The LR coffee extract had a higher antioxidant activity compared to DR under the current roasting conditions, which can be attributed to the phenolics present in the coffee extracts that underwent a lower degree of degradation in the LR processing condition. The results from the three assays on the antioxidant activity of individual fractions were, however, not always consistent. For example, Fractions III and IV showed the highest activity in ORAC assay, while Fractions II and III had the highest TEAC values. This indicates that mixtures of chemically different 68 compounds present in coffee fractions could be behaving with characteristically different antioxidant mechanisms. The formation of MRPs and the degradation of natural phenolics are two events during the roasting process of coffee which also directly relate to the final antioxidant activity of coffee. Studies by Morales’ group found that some low molecular weight compounds, particularly CGA in coffee, are ionically attached to high molecular weight melanoidins (MW>10KDa), thus contributing mostly to the antioxidant activity of coffee melanoidins (Delgado Andrade and Morales, 2005; Delgado Andrade et al. , 2005). In the present study, however, after release of the ionically attached compounds, no change or even an increased antioxidant activity of coffee melanoidins (MW>1KDa) was observed. Albeit CGA can attach to the melanoidin structures through ionic interactions, a recent study found that CGA could also be covalently bound to melanoidins (Bekedam et al. , 2008d). The later may be more predominant in the present study compared to ionic interactions. In this case, little CGA would be released from the melanoidin structures following salt treatment. Thus, no change in the antioxidant activity of coffee melanoidins was expected. In addition, low molecular weight coffee constituents that have lower antioxidant activity compared to melanoidins, or interfere the antioxidant action of melanoidins due to the ionic bonding, would be released after salt treatment, therefore, resulting in higher antioxidant activity for coffee melanoidins. A similar result has been reported for the glucose phenylalanine model MRPs, after salt treatment, where melanoidins showed a higher ABTS radical scavenging activity compared to melanoidins that had not undergone salt treatment (Rufian Henares and Morales, 2007c). In contrast, melanoidins derived from other glucose amino acid model systems showed deceased antioxidant 69 activity after salt treatment (Rufian Henares and Morales, 2007c). These inconsistent results from simple model systems indicate that the attachment of low molecular weight MRPs to the melanoidin structures has a different influence on the antioxidant activity of melanoidins, which could be either an enhanced or reduced activity. 2.4.3 Biological effects of coffee extracts The primary aim of this study was to determine if coffee brews had an effect on gastrointestinal enterocyte viability using Caco 2 cells. The MTT assay is an established method to quantify cellular growth and toxicity (Mosmann, 1983). Cells were treated for up to 72 h to achieve maximal potential toxic and biochemical effects. DR and LR coffee extracts exerted similar time and concentration dependent cytotoxic effects on Caco 2 cells. Other workers have reported that treating Caco 2 cells with drip coffee brews, prepared according to a standard procedure for 24 h, significantly reduced MTT response compared to fresh orange extract treated cells (Ekmekcioglu et al. , 1999). Associated with this observation was a 46 % reduction of cytochrome c reductase activity relatively to control (Ekmekcioglu et al. , 1999). A recent study using bovine aorta endothelial cells found that after a 24 h exposure of cells to a filter coffee and espresso coffee, the MTT cell viability reduced to 10 % and 19 % of the control, respectively (Hegele et al. , 2009). Hydrogen peroxide (H 2O2) was detected in both filter and espresso coffee brews obtained from standardized procedures, and incubation of coffee under cell culture conditions, yielded an up to 11 fold increase in H 2O2 content (Hegele et al. , 2009). These workers suggested that H 2O2 is a major product in coffee inducing cell death in vitro (Hegele et al. , 2009). 70 The effect of coffee on the antioxidant enzymes in Caco 2 cells was investigated in the present study since coffee brews exhibited high antioxidant activity in many chemical assays. A concentration of 0.1 mg freeze dried coffee/ml culture medium was used, at which no cytotoxic effect to Caco 2 cells was observed for up to 72 h. Of interest, was the result that both CAT activity and GSH content in Caco 2 cells were found to decrease after coffee treatment. CAT catalyzes the reduction of H 2O2 to water, thus normal functions of CAT and GSH are important to maintain the intracellular oxidation reduction status (redox). Redox is an important regulator of various genes that are involved in pathologically conditions, where chronic prooxidant states either initiate or exacerbate cell damage (Nath et al. , 1998; Waris and Ahsan, 2006). The inhibitory effect of coffee brew on both CAT activity and reduced GSH level could be associated with the MRPs since reduced activities of CAT and GR and GSH content has been reported in lymphocytes after MRPs treatment (Yen et al. , 2002). In addition, Int 407 cells when exposed to modeled MRPs, also showed decreased activities of SOD, CAT, GR and GPX (Jing and Kitts, 2004b). Decreased antioxidant enzyme activities and GSH content has also been reported in the blood and liver of mice that were feed methylglyoxal, an intermediate compound of the Maillard reaction (Choudhary et al. , 1997; Ankrah and Appiah Opong, 1999). Moreover, the potential of coffee and MRPs to generate ROS has been proposed by several researchers (Yen et al. , 2002; Hegele et al. , 2009), which may help explain the responses of Caco 2 cells to coffee extracts employed in this study. However, further studies are needed to elucidate how ROS are generated through Maillard reaction and how MRPs in the food system are related to either beneficial or harmful effects associated with generation or detoxification of ROS. 71 CHAPTER III COFFEE CONSTITUENTS AND MODULATION OF OXIDATIVE STATUS IN CACO-2 CELLS EXPERIMENT IIa: IN VITRO AND CELLULAR IN VITRO ANTIOXIDANT ACTIVITY OF MAILLARD REACTION PRODUCTS DERIVED FROM COFFEE EXPERIMENT IIb: IN VITRO AND CELLULAR IN VITRO ANTIOXIDANT ACTIVITY OF MAILLARD REACTION PRODUCTS DERIVED FROM SUGAR-SERINE MODEL SYSTEMS 72 3.1 Introduction Great changes in the chemical composition and biological activity of green coffee beans take place during the roasting process. These include the degradation of natural phenolic compounds, generation of Maillard reaction products (MRPs), carbohydrate caramelization, and pyrolysis of organic compounds (Guillot et al. , 1996; Belitz and Grosch, 1999). The chemical and biological properties of coffee bean constitutes derived from processing have not yet been completely elucidated. Most of the physiological effects of coffee beverages have been attributed to caffeine, the diterpenes kahweol and cafestol, and phenolic compounds (Lam et al. , 1987; Kitts and Wijewickreme, 1994; Carrillo and Benitez, 2000; Ranheim and Halvorsen, 2005). Relatively little is known about the health related effects associated with coffee MRPs due to the difficulty in isolating and recovering these compounds from coffee brews. Coffee has been shown to have strong antioxidant activity as evidenced by sequestering metal prooxidant and scavenging free radicals, which is at least partially related to MRPs (Borrelli et al. , 2002; Takenaka et al. , 2005). More recently, a low molecular weight coffee MRP, N methylpridinium was shown to have strong chemopreventive effects towards modulating Phase II enzymes both in vitro and in vivo (Somoza et al. , 2003). This finding brought a new perspective to the bioactive properties of coffee MRPs and the relationship with human health. Also, two recent studies demonstrated the presence of hydrogen peroxide (H 2O2) in roasted coffee brews and Maillard reaction model systems, and the concentration of H 2O2 was shown to increase significantly when the brew was placed in cell culture conditions (Muscat et al. , 2007; Hegele et al. , 2009). This H 2O2 could lead to nuclear translocation of the transcription factor NF κB in macrophages, 73 which may be related to an immunomodulatory effects occurring in the gut. Hydrogen peroxide has also been suggested to be the major contributor to the cytotoxicity attributed to both coffee and MRPs. However, the presence of H2O2 in coffee and MRPs could have an impact on the cellular oxidative status, which has not yet been investigated. Although the contribution of natural phenolic compounds to the antioxidant capacity of coffee cannot be ruled out, we hypothesized that MRPs contribute mostly to the antioxidant activity of coffee; with low molecular weight coffee MRPs possessing higher antioxidant activity than that of the high molecular weight MRPs. We also investigated the in vitro effects of coffee MRPs on the intracellular redox environment of human colon adenocarcinoma Caco 2 cells in which the cells were exposed to extracts generated from both green coffee, roasted coffee and model MRPs. Cellular antioxidant enzyme activities and glutathione content were measured in this regard. In particular, the expression of intestinal Caco 2 genes that are involved in human oxidative stress and antioxidant defense system were investigated. This research also evaluated possible protective effects of coffee MRPs against H2O2 induced oxidative stress in Caco 2 cells. 74 3.2 Materials and method 3.2.1 Preparation of coffee and Maillard reaction products (MRPs) 100% Coffea arabica was roasted in a commercial roaster at 204 ºC and 232 ºC for about 12 min to obtain light roasted (LR) and dark roasted (DR) coffee beans. Defatted coffee extracts were made according to methods previously described in Chapter II. Green coffee beans were treated with liquid nitrogen prior to grinding. The green bean (GB) extract was prepared the same as the roasted coffee extracts. Two model MRPs were prepared using arabinose serine (Ara Ser) and sucrose serine (Suc Ser) reactants. A solution of sugar (50 mM) and amino acid (50 mM) in distilled water (100 ml) was freeze dried and the mixtures obtained were placed into a beaker (1000 ml), and then dry heated in the oven. Based on the roasting process used for the coffee beans, two temperatures, namely 204 ºC and 232 ºC were used for LR and DR MRPs, respectively. After 12 min heating, the beakers were removed from the oven and allowed to cool to room temperature in desiccators. An aliquot (5 g) of the reaction mixture was suspended in distilled water (250 ml) and stirred for 12 min. The solution was then filtered (Whatman No.4) and the filtrate containing the water soluble MPRs was collected. The residue on the filter paper was washed with distilled water. All filtrates were collected and freeze dried to give the water soluble MRPs, which was used for the fractionation experiments. Ultrafiltration with ddH 2O was run on GB, LR and DR coffee defatted extracts, and coffee model MRPs using the same procedure as described in Chapter II. 75 3.2.2 Chemical analyses and Antioxidant assays The L, a, b and E Hunter scale color parameters and the browning of the roasted coffee extracts and fractions were measured. The UV and fluorescence spectra of both roasted coffee extracts and non roasted coffee extracts were measured. In addition, the UV and fluorescence spectra of chlorogenic acid (CGA) and tannins (Brew UK Ltd. Salisbury) were measured. The ORAC and TEAC results were calculated as Trolox equivalents (TE values). CGA was used as the reference for the RP assay. For making data comparisons, individual values were referenced to 1 gram of freeze dried sample for each of the different coffee extracts and fractions. Antioxidant capacity and reducing power measures were compared between GB, LR and DR defatted extracts in order to evaluate the effect of the roasting processes. The processes of chemical analyses and antioxidant assays were followed as described in Chapter II section 2.2.4. 3.2.2.1 Quantification of dicarbonyl compounds Dicarbonyl compounds were quantified by reversed phase HPLC after derivatization with 2,3 diaminonaphthalene (DAN; Sigma, St. Louis, MO, USA) (Chen and Kitts, 2008b). Defatted coffee extracts in 10 mM phosphate buffer (pH 7.4) were incubated with DAN in the presence of 3,4 hexanedione (Sigma, St. Louis, MO, USA) (internal standard) overnight at 4 ºC. Aqueous solutions of standard glyoxal (Sigma Aldrich, Oakville, ON, Canada), methylglyoxal (Sigma, St. Louis, MO, USA), 3 deoxyglucosone (Toronto Research Chemicals Inc. Canada), and glucosone (Sigma, St. Louis, MO, USA) 76 were treated the same way as coffee samples. The reaction mixture was then extracted by ethyl acetate and evaporated until dry under nitrogen gas. The extract was reconstituted in methanol and injected into a Sphereclone ODS2 column (Phenomenex, Torrance, CA) eluted with gradient acetonitrile (ACN) and 0.2% formic acid: 0–13 min, 28–45% ACN; 13–25 min, 45–85% ACN; 25–28 min, 85% ACN, with a flow rate of 0.8mL/min. The quinoxaline derivatives were detected by diode array detector (265 nm) and fluorescent detectors (excitation at 267 nm and emission at 503 nm). 3.2.3 Cellular in vitro Assay Caco 2 cells (passage 23 to 40) used in this chapter were cultured according to the conditions and procedures described in Chapter II. For the MTT assay, cells were seeded in 96 well plates at a density of 1 × 10 6 cells/ml 24 h before tests. For enzyme activities, glutathione content as well as real time RT PCR, cells were seeded in culture plates at a density of 2.5× 10 5 cells/ml and allowed to reach 100 % confluence before treatments. Cells were treated with extracts and fractions derived from non roasted and roasted coffee beans and model MRPs, respectively. Cells received no treatment were used as controls. Both treated and control cells were rinsed with ice cold phosphate buffer saline (PBS, pH 7.2) and scraped into a 1.5 ml tube for a freeze thaw treatment. Some cells (no more than 1× 10 6) were kept in another 1.5 ml tube for real time RT PCR. In some experiments, oxidative stress was induced by exposure of treated Caco 2 cells to 5 mM H2O2 solution in culture medium for 2 h. MTT response and cellular antioxidant enzyme activities and glutathione content were tested using the methods described in Chapter II. 77 3.2.4 Real-Time Quantitative Reverse Transcription PCR (RQ RT-PCR) Array 3.2.4.1 RNA isolation and cDNA preparation RNA was isolated using RT 2 qPCR Grade RNA Isolated Kit (PA 001, SABioscience, Frederick, MD, USA). Caco 2 cells were lysed with lysis and binding buffer and passed through a filter column to recover clear lysate before loading onto a RNA spin column for total RNA isolation. RNA was treated with RNase free DNase and suspended in RNase free H 2O. RNA quality and quantity were determined using a NanoDrop spectrophotometer (NanoDrop Technology, Wilmington, USA). All RNA samples had 260/280 ratios ≥ 2.0 and 260/230 ratio ≥ 1.7. The same amount (1.0 g) of total RNA from every sample was used for first strand cDNA synthesis using RT 2 First Strand Kit (C 03, SABioscience, Frederick, MD, USA). RNA and cDNA were stored at 80 º C. 3.2.4.2 Real time RT PCR Target mRNA was quantified on BioRad iQ5 (BioRad, USA) using RT 2Profiler™ PCR Array system (PAHS 065A, Frederick, MD, USA). This RT 2Profiler™ PCR Array contains gene specific primer sets for a thoroughly researched set of 84 genes relevant to oxidative stress and antioxidant defense system in human. There are also five housekeeping genes and three RNA and PCR quality controls (Table 3.1; also see Figure 1.4 for the layout of 96 well PCR array). RT PCR was carried out in a 96 well plate in a total volume of 25 l/well, consisting RT 2 qPCR Master Mix, first strand cDNA and pre dispensed gene specific primer. Real time PCR was performed according to the user manual: initial DNA denaturation at 95 ºC for 10 min, followed by 40 PCR cycles of denaturation at 95 ºC for 15 sec and annealing/extension 60 ºC for 1 min. Reactions were carried out in triplicate and data analysis using C t method. 78 Table 3.1 Human oxidative stress and antioxidant defense PCR array gene table Position GeneBank Symbol Description A01 NM_000477 ALB Albumin A02 NM_000697 ALOX12 Arachidonate 12 lipoxygenase A03 NM_021146 ANGPTL7 Angiopoietin like 7 A04 NM_001159 AOX1 Aldehyde oxidase 1 A05 NM_000041 APOE Apolipoprotein E A06 NM_004045 ATOX1 ATX1 antioxidant protein 1 homolog (yeast) A07 NM_004052 BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 A08 NM_001752 CAT Catalase A09 NM_002985 CCL5 Chemokine (C C motif) ligand 5 A10 NM_005125 CCS Copper chaperone for superoxide dismutase A11 NM_007158 CSDE1 Cold shock domain containing E1, RNA binding A12 NM_000101 CYBA Cytochrome b 245, alpha polypeptide B01 NM_134268 CYGB Cytoglobin B02 NM_001013742 DGKK Diacylglycerol kinase, kappa B03 NM_014762 DHCR24 24 dehydrocholesterol reductase B04 NM_175940 DUOX1 Dual oxidase 1 B05 NM_014080 DUOX2 Dual oxidase 2 B06 NM_004417 DUSP1 Dual specificity phosphatase 1 B07 NM_001979 EPHX2 Epoxide hydrolase 2, cytoplasmic B08 NM_000502 EPX Eosinophil peroxidase B09 NM_021953 FOXM1 Forkhead box M1 B10 NM_197962 GLRX2 Glutaredoxin 2 B11 NM_153002 GPR156 G protein coupled receptor 156 B12 NM_000581 GPX1 Glutathione peroxidase 1 C01 NM_002083 GPX2 Glutathione peroxidase 2 (gastrointestinal) C02 NM_002084 GPX3 Glutathione peroxidase 3 (plasma) C03 NM_002085 GPX4 Glutathione peroxidase 4 (phospholipid hydroperoxidase) C04 NM_001509 GPX5 Glutathione peroxidase 5 (epididymal androgen related protein) C05 NM_182701 GPX6 Glutathione peroxidase 6 (olfactory) C06 NM_015696 GPX7 Glutathione peroxidase 7 C07 NM_000637 GSR Glutathione reductase C08 NM_000178 GSS Glutathione synthetase C09 NM_001513 GSTZ1 Glutathione transferase zeta 1 C10 NM_001518 GTF2I General transcription factor II, i C11 NM_006121 KRT1 Keratin 1 C12 NM_006151 LPO Lactoperoxidase D01 NM_000242 MBL2 Mannose binding lectin (protein C) 2, soluble (opsonic defect) D02 NM_004528 MGST3 Microsomal glutathione S transferase 3 D03 NM_000250 MPO Myeloperoxidase D04 NM_002437 MPV17 MpV17 mitochondrial inner membrane protein 79 Table 3.1 Human oxidative stress and antioxidant defense PCR array gene table (continued) Position GeneBank Symbol Description D05 NM_012331 MSRA Methionine sulfoxide reductase A D06 NM_005954 MT3 Metallothionein 3 D07 NM_004923 MTL5 Metallothionein like 5, testis specific (tesmin) D08 NM_000265 NCF1 Neutrophil cytosolic factor 1 D09 NM_000433 NCF2 Neutrophil cytosolic factor 2 D10 NM_003551 NME5 Non metastatic cells 5, protein expressed in (nucleoside diphosphate kinase) D11 NM_000625 NOS2 Nitric oxide synthase 2, inducible D12 NM_024505 NOX5 NADPH oxidase, EF hand calcium binding domain 5 E01 NM_002452 NUDT1 Nudix (nucleoside diphosphate linked moiety X) type motif 1 E02 NM_181354 OXR1 Oxidation resistance 1 E03 NM_005109 OXSR1 Oxidative stress responsive 1 E04 NM_020992 PDLIM1 PDZ and LIM domain 1 E05 NM_015553 IPCEF1 Interaction protein for cytohesin exchange factors 1 E06 NM_007254 PNKP Polynucleotide kinase 3' phosphatase E07 NM_002574 PRDX1 Peroxiredoxin 1 E08 NM_005809 PRDX2 Peroxiredoxin 2 E09 NM_006793 PRDX3 Peroxiredoxin 3 E10 NM_006406 PRDX4 Peroxiredoxin 4 E11 NM_181652 PRDX5 Peroxiredoxin 5 E12 NM_004905 PRDX6 Peroxiredoxin 6 F01 NM_020820 PREX1 Phosphatidylinositol 3,4,5 trisphosphate dependent Rac exchange factor 1 F02 NM_006093 PRG3 Proteoglycan 3 F03 NM_183079 PRNP Prion protein F04 NM_000962 PTGS1 Prostaglandin endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) F05 NM_000963 PTGS2 Prostaglandin endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) F06 NM_012293 PXDN Peroxidasin homolog (Drosophila) F07 NM_144651 PXDNL Peroxidasin homolog (Drosophila) like F08 NM_014245 RNF7 Ring finger protein 7 F09 NM_182826 SCARA3 Scavenger receptor class A, member 3 F10 NM_203472 SELS Selenoprotein S F11 NM_005410 SEPP1 Selenoprotein P, plasma, 1 F12 NM_003019 SFTPD Surfactant protein D G01 NM_016276 SGK2 Serum/glucocorticoid regulated kinase 2 G02 NM_012237 SIRT2 Sirtuin (silent mating type information regulation 2 homolog) 2 (S. cerevisiae) G03 NM_000454 SOD1 Superoxide dismutase 1, soluble G04 NM_000636 SOD2 Superoxide dismutase 2, mitochondrial 80 Table 3.1 Human oxidative stress and antioxidant defense PCR array gene table (continued) Position GeneBank Symbol Description G05 NM_003102 SOD3 Superoxide dismutase 3, extracellular G06 NM_080725 SRXN1 Sulfiredoxin 1 homolog (S. cerevisiae) G07 NM_006374 STK25 Serine/threonine kinase 25 (STE20 homolog, yeast) G08 NM_000547 TPO Thyroid peroxidase G09 NM_003319 TTN Titin G10 NM_032243 TXNDC2 Thioredoxin domain containing 2 (spermatozoa) G11 NM_003330 TXNRD1 Thioredoxin reductase 1 G12 NM_006440 TXNRD2 Thioredoxin reductase 2 H01 NM_004048 B2M Beta 2 microglobulin H02 NM_000194 HPRT1 Hypoxanthine phosphoribosyltransferase 1 H03 NM_012423 RPL13A Ribosomal protein L13a H04 NM_002046 GAPDH Glyceraldehyde 3 phosphate dehydrogenase H05 NM_001101 ACTB Actin, beta H06 SA_00105 HGDC Human Genomic DNA Contamination H07 SA_00104 RTC Reverse Transcription Control H08 SA_00104 RTC Reverse Transcription Control H09 SA_00104 RTC Reverse Transcription Control H10 SA_00103 PPC Positive PCR Control H11 SA_00103 PPC Positive PCR Control H12 SA_00103 PPC Positive PCR Control 3.2.5 Statistical analysis Each experiment was performed in triplicate (e.g. three wells or three cell culture plates) and repeated three times in separate experiments. Collected data were expressed as mean + SD. Means were compared by One way Analysis of Variance (ANOVA), followed by Tukey’s pairwise comparisons. Comparison of different treatments with control was done by one way ANOVA, followed by Dunnett’s test. Significant differences between two samples were analyzed with Student t test. Data with two influence factors were compared by two way ANOVA with Bonferroni post tests. The level of confidence required for significance was selected at p<0.05. Statistical analyses were done by using GraphPad Prism software (version 5.01, GraphPad Software, Inc.). 81 3.3 Results Experiment IIa. 3.3.1 Recovery of coffee brews and fractions Coffea arabica (100%) was roasted in a commercial roaster at 204 ºC and 232 ºC for approximately 12 min to obtain light roasted (LR) and dark roasted (DR) coffee beans. Lyophilized coffee brew samples were defatted and separated using water ultrafiltration. The same procedure was performed on a coffee extract prepared from green coffee beans (GB), taken as a control. For each coffee sample, four fractions (I IV) were collected and the recovery for each fraction is reported in Table 3.2. GB coffee extract had greater recovery of low molecular weight components (Fraction IV) and less high molecular weight components (Fraction I), than those recovered from roasted coffee brew (p<0.05). Fraction IV contained the highest recovery of components in LR coffee, while Fraction I had the highest recovery in DR coffee. Fraction I recovered from LR and DR increased upon roasting, while the recovery decreased in Fraction IV with more roasting (p<0.05). Table 3.2 Recovery of coffee fractions by ultrafiltration 1 Fractions (MW) Recovery (% of defatted coffee dry weight) GB LR DR I (>10KDa) 15.0+0.6 ax 28.8+2.0 bcy 34.5+2.7 cy II (1 10KDa) 10.7+1.3 a 9.5+2.0 a 14.1+2.2 a III (0.5 1KDa) 25.7+3.6 b 23.5+4.3 b 20.6+7.0 ab IV (<0.5 KDa) 42.2+4.9 cy 32.6+6.8 cx 28.9+6.8 bcx 1 The values are expressed as mean + SD, n=3. abc means within the same column that do not share a common superscript letter are significantly different; xy represent significantly different means in rows. Statistical analyses using two way ANOVA with Bonferroni post tests; statistical level of confidence set as 0.05. GB = green bean extract; LR = light roasted coffee extract; DR = dark roasted coffee extract. 82 3.3.2 Chemical characteristics of coffee 3.3.2.1 Colorimetric measurements of roasted coffee Table 3.3 presents the colorimetric measurements reported on freeze dried LR and DR coffee extracts and related ultrafiltration fractions. Browning of green coffee beans occurred after roasting due to the generation of polymerized MRPs. No significant difference in colorimetric parameter L was found between LR and DR defatted coffee extracts, however, it was observed that L values of individual fractions recovered increased as the molecular weight of components that were recovered decreased. 3.3.2.2 Browning of coffee extracts An increase in browning corresponded to an increase in molecular weight of individual components (Table 3.3). This result implied a positive correlation between the degree of browning and the molecular weight of components present in derived products. No significant difference in browning was found between LR and DR. Table 3.3 Lightness (L) and browning of coffee extracts and untrafiltration fractions 1 Coffee extracts L Browning 3 LR DR LR DR Defatted brew 34.0+4.8 40.2+1.6 0.19+0.02 0.18+0.00 Fractions (MW) 2 I (>10KDa) 26.6+1.7 a 23.2+0.6 a 0.33+0.03 d 0.33+0.02 c II (1 10KDa) 37.7+0.8 b 38.3+1.0 b 0.25+0.01 c 0.25+0.04 c III (0.5 1KDa) 38.5+1.7 bx 44.2+1.4 bcy 0.17+0.03 by 0.11+0.01 bx IV (<0.5 KDa) 43.2+2.8 b 45.6+1.4 c 0.08+0.00 a 0.07+0.00 a 1 LR and DR represent light roasted and dark roasted coffee. Values represent mean + SD, n = 3. abcd The means with different superscript letters in each column are significantly different; xy represent significantly different means in rows between LR and DR (p<0.05, two way ANOVA with Bonferroni post tests). 2 Fractions are derived from defatted extracts, based on molecular weight (MW). 3 Browning intensities are absorbance reading at 420 nm. Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 83 3.3.2.3 Fluoresence and UV visible spectra The GB, LR and DR coffee defatted extracts were also compared for relative difference in fluorescence spectra (Figure 3.1). A sharp, distinct peak appearing at 400 nm emission wavelength was observed in the GB coffee defatted extract and related fractions possessed (Figure 3.1B). Fraction IV recovered from GB defatted extract had a small peak occurring at 400 nm. The other three fractions recovered from the GB extract also had very high intensity peaks (Not shown in the Figure in order to avoid high overlapping). These peaks were summarized as follows: FI Fraction I : 1020; FI Fraction II : 860; FI Fraction III : 1010 (where FI = fluorescent intensity). To verify that these peaks were related to natural phenolics, a fluorescence spectrum for chlorogenic acid (CGA) and tannins (0.05 mg/ml) was also measured. The concentration of CGA was based on an estimated proportion (e.g. 20 %) in soluble GB extracts (Farah and Donangelo, 2006; Arya and Rao, 2007). The fluorescence spectra of 0.05 mg/ml CGA exhibited a sharp peak at 400 nm emission wavelength (Figure 3.1A). However, the intensity of this peak (≈120) was much lower than the peak obtained from the GB coffee extracts. The peak of tannins (FI = 400) at 400 nm emission wavelength had an intensity that was relatively higher than CGA. This result indicated that high molecular weight polyphenolics are present in green coffee beans extract, and possessed components with a characteristic spectra pattern observed at a 400 nm emission wavelength. Fraction I recovered from both LR and DR coffee also contained a sharp fluorescent peak at 400 nm, and another broader peak at a higher emission wavelength (Figure 3.1D). The intensity of the peak at 400 nm emission wavelength from the roasted coffee was much lower than that derived from the GB coffee, likely indicating a loss of phenolic compounds during the roasting 84 process. The peaks at 400 nm present in Fractions II, III and IV recovered from roasted coffee had relatively lower intensities compared to the broad peaks that occurred between 450 nm to 500 nm in these same fractions (Figure 3.1 E G). This finding supports the possibility that greater amounts of MRPs were presented in these three fractions than phenolics after roasting of the coffee beans. The pattern of the fluorescence spectra for roasted coffee extracts shown here are similar to the fluorescent spectra for the roasted coffee extracts in Chapter II (Figure 2.1). Figure 3.2 shows the UV spectra of GB, LR and DR coffee defatted extracts and ultrafiltration fractions over an absorbance range of 250 700 nm. Both LR and DR defatted coffee extracts had peak absorbance at around 275 nm, with a shoulder occurring at 320 nm (Figure 3.2B). The UV absorbance spectra of Fraction I recovered from LR and DR coffee extracts decreased continuously with increasing wavelength. In contrast, the absorbance spectra for Fractions II and III recovered from LR and DR coffee extracts had absorption peaks at 275 nm, with shoulders occurring at longer wavelengths. Fraction IV also had a sharp peak at 275 nm. GB coffee samples had different UV spectral patterns compared to roasted coffee samples (Figure 3.2 B F). The UV absorbance spectrum of 0.1 mg/ml CGA was also tested (Figure 3.2A). As expected, CGA showed a similar UV spectral pattern as observed with the GB coffee extract. These results indicate that GB contained relatively higher amounts of CGA, which greatly influenced the UV spectral pattern of GB coffee extract. However, these phenolic compounds were altered during roasting, resulting in products that did not possess a similar UV spectral pattern. 85 Figure 3.1 Fluorescence emission spectra (350 550 nm) of green bean (GB), light roasted (LR) and dark roasted (DR) coffee extracts and fractions. The fluorescent spectra of 0.05 mg/ml CGA ( ) and tannins ( ) are shown in (A). The spectra of GB defatted extract ( ) and Fraction IV ( ) are shown in (B). LR ( ) and DR ( ) defatted coffee extracts (C), Fraction I (D), Fraction II (E), Fraction III (F) and Fraction IV(G). All emission spectra were measured with the excitation wavelength set at 400 nm. Samples were diluted to 0.25 mg/ml with mili Q water prior to spectrum measurement. 86 Figure 3.2 Comparison of the UV visible spectra of green bean (GB), light roasted (LR) and dark roasted (DR) coffee extracts and fractions. The UV spectra of 0.1 mg/ml CGA ( ) and tannins ( ) are shown in (A). The spectra of GB ( ), LR ( ) and DR ( ) are defatted coffee extracts (B), Fraction I (C), Fraction II (D), Fraction III (E) and Fraction IV(F). Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 87 3.3.2.4 Characterization of coffee α dicarbonyl compounds Derivatization of α dicarbonyl compounds occurring in defatted coffee extracts was performed using 2,3 diaminonaphthalene derivatization at 4 ºC overnight. Aqueous solutions of standard glyoxal, methylglyoxal, 3 deoxyglucosone, and glucosone were also derivatized and 3, 4 hexanedione was added as the internal control. Glyoxal and methylglyoxal were identified in both defatted LR and DR coffee extracts. The concentrations of these two specific α dicarbonyl are shown in Figure 3.3; methylglyoxal being the predominant α dicarbonyl compound recovered from both LR and DR coffee, respectively. Methylglyoxal concentration in LR coffee was significantly higher (p<0.05) than that in DR coffee (p<0.05), while there was no significant difference in the glyoxal content between LR and DR coffee. Figure 3.3 Alpha dicarbonyl compounds in light roasted (LR: ) and dark roasted (DR: ) coffee extracts. * indicates significant difference between LR and DR (Student t test, p<0.05). 3.3.3 Antioxidant activity of roasted and green coffee The antioxidant capacity and reducing power of GB, LR and DR coffee extracts and ultrafiltration fractions were measured by ORAC, TEAC and RP assays (Table 3.4 3.7). 88 Table 3.4 presents the affinity of GB, LR, and DR coffee to scavenge peroxyl radicals and ABTS•+ radicals, respectively, and corresponding reducing activity. The antioxidant indicator values for ORAC, TEAC and RP for GB defatted extract were significantly higher (p<0.05) than those obtained from LR and DR coffee. Data from the ORAC and TEAC assays suggested that the free radical scavenging activity decreased with increased roasting processing of coffee beans. Table 3.4 Antioxidant activity of coffee extracts 1 Coffee Assays 2 ORAC TEAC RP GB 2.50+0.09 c 0.63+0.01 c 0.31+0.02 b LR 1.39+0.07 b 0.53+0.02 b 0.16+0.00 a DR 1.12+0.06 a 0.45+0.01 a 0.15+0.01 a 1 Value represents mean + SD (n=3); abc represent significant different means in columns (p<0.05, one way ANOVA with Tukey’s pairwise comparisons). GB = green bean extract; LR = light roasted coffee extract; DR = dark roasted coffee extract. 2 ORAC represents oxygen radical absorption capacity; TEAC represents Trolox equivalent antioxidant capacity; RP represent reducing power. ORAC and TEAC values are expressed as mmol Trolox equivalents/g freeze dried coffee samples; RP values are expressed as mg CGA/g freeze dried coffee samples. Table 3.5 presents the ORAC values for GB, LR, and DR coffee fractions. Fraction III recovered from both GB and the LR coffee defatted extracts exhibited the highest antioxidant activity against generated peroxyl radicals (p<0.05). Fractions II and III recovered from DR coffee beans also showed higher ORAC activity compared to Fractions I and IV (p<0.05). The ORAC values of GB fractions were significantly (p<0.05) higher in comparison to the corresponding fractions recovered from roasted coffee. Distinct from ORAC results, Fractions II and III recovered from GB had the highest (p<0.05) TEAC values. Fraction II from both LR and DR coffee had the highest 89 (p<0.05) TEAC value (Table 3.6). Fractions II, III and IV recovered from GB had higher TEAC values than those recovered from LR and DR coffee, respectively, while the TEAC of LR Fraction I was higher than that of the GB Fraction I (p<0.05). Table 3.7 presents the ferric reducing power of coffee fractions. Among GB fractions, the RP of Fractions II and III were significantly higher (p<0.05) than that of Fractions I and IV. No significant difference in RP was found among LR fractions. For DR coffee, Fraction II had the highest RP value, but this was not significantly different from Fractions I and III. The RP values of GB coffee fractions were significantly higher (p<0.05) than those of roasted coffee fractions. Table 3.5 Antioxidant activity of coffee fractions determined by the ORAC method 1 Fractions (MW) 2 ORAC (mmol TE/ g sample) GB LR DR I (>10 KDa) 2.13+0.16 ay 1.31+0.01 ax 1.11+0.10 ax II (1 10 KDa) 2.51+0.11 az 1.67+0.01 by 1.39+0.06 bx III (0.5 1 KDa) 3.38+0.19 bz 1.98+0.02 cy 1.45+0.06 bx IV (<0.5 KDa) 2.37+0.27 az 1.38+0.03 ay 1.03+0.08 ax 1 ORAC represents oxygen radical absorption capacity. Values are expressed as mmol Trolox equivalents/g freeze dried coffee samples (mean + SD, n=3). abc means within the same column that do not share a common superscript letter are significantly different; xyz represent significantly different means in rows. Statistical analyses using two way ANOVA with Bonferroni post tests; statistical level of confidence set at 0.05. GB = green bean extract; LR = light roasted coffee extract; DR = dark roasted coffee extract. 2 Fractions were derived from defatted coffee extracts, based on molecular weight (MW). 90 Table 3.6 Antioxidant activity of coffee fractions determined by the TEAC method 1 Fractions (MW) 2 TEAC (mmol TE/ g sample) GB LR DR I (>10 KDa) 0.44+0.03 ax 0.56+0.02 by 0.50+0.02 bcxy II (1 10 KDa) 0.84+0.01 cz 0.73+0.01 dy 0.56+0.02 cx III (0.5 1 KDa) 0.80+0.03 cz 0.66+0.03 cy 0.46+0.03 bx IV (<0.5 KDa) 0.68+0.07 bz 0.48+0.05 ay 0.39+0.02 ax 1 TEAC represents Trolox equivalent antioxidant capacity. Values are expressed as mmol Trolox equivalents/g freeze dried coffee samples (mean + SD, n=3). abc means within the same column that do not share a common superscript letter are significantly different; xyz represent significantly different means in rows. Statistical analyses using two way ANOVA with Bonferroni post tests; statistical level of confidence set as 0.05. GB = green bean extract; LR = light roasted coffee extract; DR = dark roasted coffee extract. 2 Fractions were derived from defatted extracts, based on molecular weight (MW). Table 3.7 Antioxidant activity of coffee fractions determined by the RP method 1 Fractions (MW) 2 RP (mmol CGA/ g sample) GB LR DR I (>10 KDa) 0.20+0.04 ay 0.17+0.01 x 0.17+0.01 abx II (1 10 KDa) 0.43+0.05 cy 0.22+0.02 x 0.21+0.02 bx III (0.5 1 KDa) 0.42+0.05 cy 0.19+0.01 x 0.15+0.01 abx IV (<0.5 KDa) 0.35+0.05 by 0.16+0.01 x 0.14+0.01 ax 1 RP represents reducing power. Values are expressed as mg CGA/g freeze dried coffee samples (mean + SD, n=3). abc means within the same column that do not share a common superscript letter are significantly different; xy represent significantly different means in rows. Statistical analyses using two way ANOVA with Bonferroni post tests; statistical level of confidence set as 0.05. GB = green bean extract; LR = light roasted coffee extract; DR = dark roasted coffee extract. 2 Fractions were derived from defatted extracts, based on molecular weight (MW). 3.3.4 Biological effects of coffee bean extracts 3.3.4.1 MTT response The MTT assay is based on the reduction of the tetrazolium ring of MTT by mitochondrial dehydrogenases yielding a purple formazan product, which is measured spectrophotometrically. The amount of formazan produced is proportional to the number 91 of viable cells. Caco 2 cells were exposed to defatted extracts derived from GB, LR and DR coffee samples with a concentration ranging from 0.005 to 10 mg freeze dried extract/ ml culture medium. Cell response curves are presented in Figure 3.4 and the IC50 of Caco 2 cells after treatment with GB, LR, and DR coffee defatted extracts for different time periods are summarized in Table 3.9. Both LR and DR coffee extracts reduced the viability of Caco 2 cells in a time and concentration dependent manner. GB extract did not show the same cytotoxicity as roasted coffee extracts. Significantly different (p<0.05) IC50 were obtained for GB coffee treated cells, compared to cells treated with LR and DR coffee. For all time periods, the IC50 values of roasted coffee treated cells were lower than cells treated with GB coffee (p<0.05). There was no significant difference in IC50 values between cells treated with LR and DR coffee extracts. 1 Table 3.9 IC 50 of coffee extracts on Caco 2 cells using MTT assay Time IC 50 (mg/ml) GB LR DR 3h 16.60+1.91 cx 13.57+1.30 cx 12h 21.81+1.17 ay 7.01+0.26 bx 6.98+0.35 bx 24h 22.28+1.34 ay 5.72+0.37 abx 5.28+0.13 ax 48h 31.73+1.25 cy 4.73+0.19 abx 4.54+0.14 ax 72h 26.95+1.19 by 3.99+0.16 ax 3.75+0.10 ax 1 IC 50 is determined as the concentration of coffee that is required to reduce cell viability to half that of control in the MTT assay. Values represent means + SD obtained for triplicate samples measured in triplicate. abcd represent means in columns that are significantly different; xy represent significantly different means in rows. Statistical analyses were done by one way ANOVA with Tukey’s pairwise comparisons; level of confidence set as 0.05. GB = green beans; LR = light roast; DR = dark roast. 92 Figure 3.4 Effects of green bean (GB), dark roasted (DR) and light roasted (LR) coffee extracts on the tetrazolium reduction rate in the MTT assay after 3 h ( ), 12 h ( ), 24 h ( ), 48 h ( ) and 72 h ( ) incubation. (A): GB; (B): LR; (C): DR Data (% control) represent means + SD obtained for triplicate samples measured in triplicate. 93 3.3.4.2 Caco 2 cellular glutathione content after coffee extracts treatment All types of coffee used in this study showed high radical scavenging activity and reducing capacity in the chemical antioxidant assays. However, the effects of coffee extracts in the biological system used herein produced different results. It has been reported that Caco 2 cells exhibited antioxidant mechanisms mainly through the glutathione (GSH) cycle, a principle system that involves the adaptation to, and prevention of, cell oxidative damage (Baker and Baker, 1993). Considering the role of GSH as one of the most important intracellular defenses, the GSH level in Caco 2 cells exposed to 1.0 mg/ml of GB, LR and DR coffee defatted extracts for up to 72 h was measured (Figure 3.5). The concentration of coffee extracts selected from the MTT response was based on preliminary evidence that showed no cytotoxic effect at this concentration. The concentrations of 1.0 mg/ml and 2.0 mg/ml have been commonly used by other researchers to look at the cellular effects of coffee and other MRPs containing food (Somoza et al. , 2003; Jing and Kitts, 2004b; Muscat et al. , 2007). There was no significant difference in GSH content, quantified in cultures which were exposed to GB, LR, and DR coffee extracts, respectively, for 3 h, compared to control. In contrast, cells treated with LR and DR coffee extracts, respectively, for 24 h and 72 h, exhibited a decreased intracellular GSH content relative to control (p<0.05), while no significant change in GSH content was found in cells treated with GB extract. 94 Figure 3.5 Effect of coffee extracts on glutathione (GSH) content in Caco 2 cells. Cells were exposed to1.0 mg/ml green bean (GB), light roasted (LR) and dark roasted (DR) coffee for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. * indicates significant difference compared to corresponding control (p<0.05). Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. 95 3.3.4.3 Caco 2 antioxidant enzymes following exposure to coffee extracts It is proposed that antioxidant enzymes are primarily responsible for the change in intracellular antioxidant status and are fairly sensitive biomarkers of cellular oxidative stress and activity of the antioxidant defense system. As shown in Figure 3.6, the activity of glutathione peroxidase (GPX) was significantly decreased in Caco 2 cells (p<0.05) after 3 h exposure to 1.0 mg/ml LR and DR coffee defatted extracts. However, the activity of GPX increased significantly (p<0.05) after 24 h LR coffee exposure. Increased GPX activity was also observed in DR coffee treated cells, but this change was not significant. Increased GPX activity in Caco 2 cells could partially explain the reduced GSH content observed at 24 h after LR coffee treatment, since increased GPX activity will accelerate the oxidation of GSH to GSSG. However, the reduction of GSSG in cells was limited due to the unchanged, or even slightly reduced, GR activity observed after 24 h of LR coffee treatment (not significant, p>0.05, data presented in Appendix Table 4). After 72 h of LR coffee treatment, both GPX and CAT activity in Caco 2 cells were significantly decreased (p<0.05), when compared to the control groups. The decreased CAT activity was observed after 24 h treatment with LR and DR coffee extracts (p<0.05). GB coffee treatments produced no stimulatory, or inhibitory, effects on the cellular antioxidant enzyme activities for up to 72 h exposure. This finding indicates a possible involvement of coffee MRPs in the regulation of antioxidant enzyme activity in Caco 2 cells. Reduced superoxide dismutase (SOD) activities were observed in Caco 2 cells exposed to coffee extracts, but the changes were not found to be significant (data in Appendix Table 5). 96 Figure 3.6 Effect of coffee extracts on glutathione peroxidase (GPX) activity in Caco 2 cells. Cells were exposed to1.0 mg/ml green bean (GB), light roasted (LR) and dark roasted (DR) coffee for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. * indicates significant difference compared to corresponding control (p<0.05). Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. 97 Figure 3.7 Effect of coffee extracts on catalase (CAT) activity in Caco 2 cells. Cells were exposed to1.0 mg/ml green bean (GB), light roasted (LR) and dark roasted (DR) coffee for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. * indicates significant difference compared to corresponding control (p<0.05). Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. 98 3.3.5 Biological effects of coffee fractions on Caco-2 cells Fractions II and III recovered from GB and LR coffee defatted extracts were chosen in this experiment in order to gain further insight into the biological effects of coffee containing components. Two different experimental designs were used: (1) treatment of cells with 1.0 mg/ml coffee extracts and fractions, respectively, to test for a direct effect; (2) pretreatment of cells with 1.0 mg/ml coffee extracts and fractions, respectively, before exposing the cells to an oxidative stress using 5 mM H 2O2 for 2 h. Cell viability, GSH content, and antioxidant enzyme activities were evaluated in both experiments. 3.3.5.1 MTT response Cellular MTT responses to GB and LR coffee samples, including defatted extracts, Fractions II and III, both with (+) or without ( ) H2O2 treatment are summarized in Table 3.10. After 24 h treatment of Caco 2 cells with Fraction II recovered from LR coffee, a significant decrease (p<0.05) in cell viability was observed compared to that of the negative control, which contained Caco 2 cells grown in culture medium without H2O2 treatment. No significant cytotoxic effect on Caco 2 cells was found for other GB and LR coffee samples after 3 h incubation. Increasing the exposure time of cells to coffee samples to 24 h, did not produce any further significant stimulatory or inhibitory effect on cellular MTT response. Exposure of Caco 2 cells to H 2O2, reduced cell viability to 80 % of negative control, and this effect was not prevented by pre incubation of cells with either GB or LR coffee samples for 3 h or 24 h, respectively. The pretreatment of cells with defatted extract and Fraction II derived from LR coffee resulted in a greater degree of Caco 2 cytotoxicity, compared to the positive control (+H2O2). Cell viability decreased to 63 % and 58 % of 99 negative control, respectively. In general, these results indicated that exposure of Caco 2 cells to GB and LR coffee was not cytotoxic. The exception to this was when cells were treated with Fraction II derived from LR coffee for 24 h. Moreover, these coffee treatments of Caco 2 cells did not produce a protective effect against H 2O2 induced cytotoxicity. In fact, exposure of cells to the defatted extract and Fraction II derived from LR coffee may have further enhanced the cytotoxic effects of H 2O2. 1 Table 3.10 Caco 2 MTT response to coffee with (+) and without ( ) H 2O2 treatment H 2O2 + H 2O2 Control 2 100+6 80+4* 3 h coffee Treatment GB H 2O2 + H 2O2 Defatted 101+3 90+6 Fraction II 110+1 93+3 Fraction III 93+5 92+2 LR Defatted 98+4 79+7 Fraction II 98+7 73+1 Fraction III 94+2 86+7 24 h coffee Treatment GB H 2O2 + H 2O2 Defatted 104+2 69+3 Fraction II 107+6 72+5 Fraction III 110+4 72+7 LR Defatted 92+1 63+2** Fraction II 80+4* 58+2** Fraction III 95+8 77+4 1 Data (% control) represent means + SD obtained for triplicate samples measured in triplicate. Significant changes are expressed as * (p<0.05) in comparison with negative ** control ( H2O2), and (p<0.05) in comparison with positive control (+ H2O2) (one way ANOVA followed by Dunnett’s multiple comparison tests). 2 The value of positive control (+ H2O2) was significantly (p<0.05) lower than that of negative control ( H2O2). 100 3.3.5.2 Caco 2 Cellular glutathione content following exposure to coffee extracts The concentration of GSH was measured in Caco 2 cells treated with defatted extracts, Fractions II and III, respectively, derived from GB and LR coffee for 24 h. GSH content was also measured in these cells which were subsequently treated with H 2O2 for 2 h. As a positive control to stimulate a prooxidant effect on changes in GSH utilization, cells were exposed to only H2O2 for 2 h and the results are shown in the same figure (Figure 3.8). Significant decreases (p<0.05) in GSH content were observed in cells treated with defatted LR coffee extract and the recovered Fractions II, respectively, compared to the negative control. No significant change in GSH content was found in Caco 2 cells after GB sample treatment. Exposure of Caco 2 cells to GB and LR coffee samples did not prevent H2O2 induced reduction of cellular GSH content. Figure 3.8 Effect of light roasted (A) and green bean (B) coffee extracts on Caco 2 cellular GSH contents with (+: ) and without ( : ) H 2O2 treatment. Caco 2 cells treated with coffee samples alone were compared to negative control ( H2O2), and cells pretreated with coffee samples and further challenged by H2O2 were compared to positive control (+ H2O2). *represents significant difference in comparison with negative contol. There was no significant change in GSH content in cells that were pretreated with coffee samples and further challenged by H2O2 compared to that of positive control. Statistical analyses were done by one way ANOVA with Dunnett’s tests (p<0.05). 101 3.3.5.3 Caco 2 antioxidant enzyme activities following exposure to coffee extracts The presence of H 2O2 in the culture medium induced a significant increase (p<0.05) in the enzyme activity of GPX compared to negative control (Figure 3.9 AB). Treating Caco 2 cells with the LR coffee extract alone, also increased GPX activity (p<0.05) (Figure 3.9 A). The GPX induction was not observed when cells were treated with either Fraction II or III derived from the LR coffee. No significant change in GPX activity was observed for cells treated with GB coffee samples alone (Figure 3.9 B). The GPX activity in cells pretreated with LR coffee samples was not significantly different compared to the positive control. However, cells pretreated with GB samples showed significantly (p<0.05) decreased GPX activity compared to the positive control, indicating the prevention of H2O2 induced GPX activity when pre exposed to GB extracts. Exposure of Caco 2 cells to H 2O2 resulted in a reduced cellular GR activity in the present study (Figure 3.9 CD). Cells pretreated with LR coffee Fraction III exhibited a significant decrease in the GR activity, compared to the positive control (p<0.05). No significant changes in GR activity were found in all other samples when treated with coffee extracts alone, or when producing a further induction in oxidative stress. As shown in Figure 3.9 E and F, respectively, the presence of H 2O2 for 2 h resulted in a significant increase in CAT activity in Caco 2 cells (p<0.05). A pretreatment of cells with LR coffee samples prevented the H2O2 induced increase in CAT activity. Treatment of Caco 2 cells with either Fraction II or III derived from LR coffee had no effect on cellular CAT activity. There was no significant difference in Caco 2 CAT activity between GB treated cells and corresponding control cells (p>0.05). 102 Caco 2 SOD activity was not significantly affected in any of the different experimental conditions or treatments used above (data in Appendix Table 6 7). 103 Figure 3.9 Effect of light roasted (LR) and green bean (GB) coffee extracts on Caco 2 cellular antioxidant enzyme activities with ( ) and without ( ) H 2O2 treatment. A: GPX activity, LR treatment; B: GPX activity, GB treatment; C: GR activity, LR treatment; D: GR activity, GB treatment; E: CAT activity, LR treatment; F: CAT activity, GB treatment. Cells treated with coffee samples alone were compared to negative control ( H2O2), and cells pretreated with coffee samples and further challenged by H2O2 were compared to positive control (+H2O2). *indicates significant difference in comparison with negative control, and ** indicates significant difference in comparison with positive control. Statistical analyses were done by one way ANOVA with Dunnett’s tests (p<0.05). 104 3.3.6 The regulatory effects of coffee on the expression of the genes involved in the oxidative stress and antioxidant defense system in Caco-2 cells Treating Caco 2 cells with LR and DR defatted coffee extracts for 24 h resulted in both an over and under expression of numerous genes that are involved in the human oxidative stress and antioxidant defense system. Table 3.11 summarizes the gene expression responses obtained from Caco 2 cells treated with GB, LR, and DR coffee extracts and hydrogen peroxide for 24 h; the latter of which was used as a positive control. Of the three different coffee extracts tested, the one derived from green coffee beans, was used as the negative control. Changes in gene expression are expressed as a fold change, which is the ratio of the gene expression between treated and untreated conditions. A significant change in gene expression was denoted as a fold change greater than 2 fold (up or down) (p<0.05). Exposure of Caco 2 cells to GB coffee extract resulted in a significant (p<0.05) increase in GPX2 and a decrease (p<0.05) in MT3 expression. This result compared to 9 genes that were significantly affected when cells were exposed to H 2O2 (p<0.05). Caco 2 cells exposed to the DR coffee extract had 13 specific genes where expression was significantly altered (P<0.05). LR coffee extract exhibited the greatest influence on Caco 2 cell gene expression, with 11 genes being up regulated (p<0.05). Relative changes in gene expression after exposure to LR coffee extract were found to be greatest for GPX2 (+18.74 fold) and iNOS (+11.46 fold). Examples of genes that were down regulated, included NME5 ( 9.46 fold), ALB ( 6.72 fold), PRG3 ( 6.00 fold), SEPP1 ( 5.73 fold), and MT3 ( 5.21 fold). Descriptions of these genes and relative function are given in the Appendix Table 8. Genes containing antioxidant response elements (ARE) in the 105 promoter region are of particular interest in the present study. The regulation of these genes by LR coffee extract, DR coffee extract, and H 2O2, respectively, are shown in the Figure 3.10. Overall, treatment of Caco 2 cells with the LR coffee extract resulted in greater changes in gene expression, both in the number of genes and more so in the magnitude of change when compared to cells exposed to either DR coffee extract or H2O2, respectively. For example, GPX2 was up regulated 18.74 fold by LR coffee treatment and 13.88 fold by DR coffee treatment. In contrast, GPX2 expression was up regulated 2.43 fold and 3.68 fold, when cells were treated with GB coffee (negative control) and H2O2 (positive control), respectively. iNOS expression increased significantly (p<0.05) in Caco 2 cells treated with LR and DR coffee extracts and H2O2, respectively, but not in cells treated with GB extract. The gene regulatory effects of LR, DR and GB coffee extracts and H2O2 on the expression of GPX2 , iNOS , SRXN1 , TXRD1 , PRDX4 and CAT in Caco 2 cells fell into the same order: LR > DR > H 2O2 > GB; where cells treated with LR coffee extracts had the greatest change in these three gene expression, followed by cells treated with DR coffee extract and H2O2, respectively. No significant change occurred in the expression of these six genes when cells were treated with GB coffee extract. 106 Table 3.11 Genes differently expressed in Caco 2 cells after incubation with coffee 1 extracts and H 2O2 Gene symbol Treatment GB LR DR H2O2 GPX2 +2.43 +18.74 +13.88 +3.68 iNOS n/c +11.46 +7.10 +2.58 SRXN1 n/c +5.00 +2.79 n/c NCF2 n/c +2.88 n/c n/c TXNRD1 n/c +2.84 +2.07 n/c PXDN n/c +2.79 n/c n/c PRDX4 n/c +2.46 +2.00 n/c MBL2 n/c +2.45 +2.64 n/c SELS n/c +2.29 n/c n/c PRDX1 n/c +2.10 n/c n/c PRDX6 n/c n/c n/c +2.00 NME5 n/c 9.46 n/c 2.42 ALB n/c 6.72 2.97 5.66 PRG3 n/c 6.00 2.75 n/c SEPP1 n/c 5.73 4.22 2.20 NOX5 n/c 5.25 4.95 7.77 MT3 2.85 5.21 2.15 4.11 CAT n/c 3.82 2.49 n/c PREX1 n/c 3.03 n/c n/c ATOX1 n/c 2.59 n/c n/c MSRA n/c 2.35 2.52 n/c AOX1 n/c 2.28 n/c 5.65 PTGS1 n/c 2.26 n/c n/c 1 Data represent fold changes of differently expressed genes in treated cells compared to control cells (n=3, fold change>2, p<0.05). “+” = up regulation; “ ” = down regulation; n/c = no significant change (p>0.05) or fold change<2. The name and function of these genes are described in Appendix (Table 8). 107 Figure 3.10 Antioxidant genes expression in Caco 2 cells treated with light roasted (LR: ), dark roasted (DR: ) coffee extracts and H 2O2 ( ) compared to those in control cells. A: up regulated gene; B: down regulated gene. *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05. GPX2 = glutathione peroxidase 2; SRXN1 = sulfiredoxin 1; TXNRD1 = thioredoxin reductase 1; PRDX4 = peroxiredoxin 4; PRDX1 = peroxiredoxin 1; PRDX6 = peroxiredoxin 6; CAT = catalase. 108 EXPERIMENT IIb 3.3.7 Chemical characteristics and antioxidant activity of Suc-Ser and Ara-Ser model MRPs Studies have indicated that serine and threonine represent two amino acids that react with sugars, such as arabinose and sucrose to produce MRPs during the roasting process of green coffee beans (De Maria et al., 1996; Reichardt et al., 2009). In this experiment, arabinose serine (Ara Ser) and sucrose serine (Suc Ser) MRPs were prepared according to the same roasting condition used to obtain LR and DR coffee beans, characterized earlier in Experiment II. The chemical characteristics and antioxidant activity associated with these two types of model MRPs were investigated in order to choose the appropriate model MRPs to represent MRPs derived from coffee beans. 3.3.7.1 Colorimetric measurements and browning of Suc Ser and Ara Ser model MRPs Table 3.12 presents data of lightness (L) and browning obtained from Suc Ser and Ara Ser model MRPs. L values for LR and DR Ara Ser MRPs were lower (p<0.05) than the corresponding L values for Suc Ser MRPs. These findings indicated greater darkness generated from heat processed Ara Ser reactions. Similarly, Ara Ser MRPs had a significantly higher (p<0.001) degree of browning, compared to Suc Ser MRPs when processed under identical conditions. There was no significant different in L values and browning between MRPs derived from LR and DR processes. 109 Table 3.12 Lightness (L) and browning of model MRPs 1 MRPs samples L Browning 2 LR DR LR DR Suc Ser MRPs 26.2+2.1 b 25.5+1.8 b 0.25+0.04 a 0.31+0.02 a Ara Ser MRPs 17.6+1.7 a 20.2+0.7 a 0.56+0.01 b 0.54+0.03 b 1 LR and DR represent light roasted and dark roasted MRPs. Values represent mean + SD, n = 3. ab The means with different superscript letters in each column are significantly different (p<0.05, two way ANOVA with Bonferroni post tests). 2 Browning intensities are absorbance reading at 420 nm. Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 3.3.7.2 Fluorescence and UV spectra of Suc Ser and Ara Ser model MRPs Figure 3.11 shows the different fluorescence intensities obtained for Suc Ser and Ara Ser model MRPs. The emission spectra, with maximum emission wavelength at 470 nm for Suc Ser MRPs and Ara Ser MRPs crude extracts were both obtained at an excitation wavelength at 400 nm. The maximum fluorescence intensity of DR MRPs derived from the Suc Ser model system was relatively greater (p<0.05) than that of the LR MRPs. There was no significant difference in maximum fluorescence intensity between LR and DR MRPs derived from the Ara Ser model system. However, LR MRPs derived from the Ara Ser system had a peak that occurred at 400 nm emission wavelength, where the DR Ara Ser MRPs had only little fluorescence. Figure 3.12 shows the different UV spectra obtained for Suc Ser and Ara Ser model MRPs, respectively. Suc Ser and Ara Ser MRPs crude extracts had similar characteristic peak absorbance at 285 nm, with the DR model systems showing relatively higher peak absorbance than the LR ones. 110 Figure 3.11 Fluorescence emission spectra (350 550 nm) of light roasted (LR ) and dark roasted (DR ) Sugar Serine MRPs extracts. Suc Ser MRPs extracts (A), and Ara Ser MRPs extracts (B), measured with the excitation wavelength set at 400 nm. Samples were diluted to 0.25 mg/ml with Milli Q water prior to spectrum measurement. Figure 3.12 UV spectra of light roasted (LR ) and dark roasted (DR ) Sugar Serine MRPs extracts. Suc Ser MRPs extracts (A), and Ara Ser MRPs extracts (B), measured over a range of absorbance wavelength (250 700 nm). Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 111 3.3.7.3 Generation of α dicarbonyl compounds in model MRPs Two α dicarbonyl compounds, glyoxal and methylglyoxal, were identified in both the LR and DR Ara Ser MRPs (Figure 3.13 A). There was no significant difference in the relative concentrations of α dicarbonyls between LR and DR MRPs derived from the Ara Ser model system. Unlike Ara Ser MRPs, four α dicarbonyl compounds, namely glyoxal, methylglyoxal, 3 deoxyglucosone, and glucosone, were identified in the Suc Ser model MRPs (Figure 3.13 B). The concentration of 3 deoxyglucosone was significantly higher (p<0.05) than that of the other three α dicarbonyl compounds, in both LR and DR Suc Ser MRPs. The concentrations of glyoxal and methylglyoxal were higher in DR Suc Ser MRPs compared to those of LR Suc Ser MRPs, respectively (p<0.05). In contrast, the glucosone concentration was higher in the LR Suc Ser MRPs (p<0.05). In general, the concentration of total α dicarbonyl compounds in Suc Ser MRPs was greater (p<0.05) than that in Ara Ser MRPs. This was primarily due to the high 3 deoxyglucosone content in Suc Ser MRPs. 114 Figure 3.13 Alpha dicarbonyl compounds in Ara Ser MRPs (A) and Suc Ser MRPs (B). Light roasted (LR: ); dark roasted (DR: ) MRPs crude extracts. DOG = 3 deoxyglucosone; GLO = glyoxal; MGL = methylglyoxal; GLS = glucosone. * indicates significant difference between LR and DR (Student t test, p<0.05). 115 3.3.7.4 Antioxidant activity of model MRPs Table 3.13 summarizes the antioxidant activity and the reducing power measured from both the Ara Ser and Suc Ser MRPs crude extracts, using the ORAC, TEAC and the RP assay. Generally, Ara Ser MRPs had relatively higher antioxidant activity compared to Suc Ser MRPs (p<0.05). Table 3.13 Antioxidant activity of Suc Ser and Ara Ser MRPs 1 MRPs Assays 2 ORAC TEAC RP LR DR LR DR LR DR Suc Ser 0.49+0.01 a 0.45+0.06 a 0.16+0.01 a 0.19+0.01 a 0.15+0.01 a 0.17+0.01 a Ara Ser 1.06+0.17 b 0.96+0.12 b 0.65+0.03 b 0.57+0.04 b 0.21+0.02 b 0.24+0.03 b 1 LR and DR represent light roasted and dark roasted model MRPs. Value represents mean + SD (n=3). ab represent significantly (p<0.05) different means in each column; no significant difference between LR and DR (p>0.05). Significant differences were analyzed using two way ANOVA with Bonferroni post tests. 2 ORAC represents oxygen radical absorption capacity; TEAC represents Trolox equivalent antioxidant capacity; RP represents reducing power. ORAC and TEAC values are expressed as mmol Trolox equivalents/g freeze dried MRPs samples; RP values are expressed as mg CGA/g freeze dried MRPs samples. MRPs derived from Ara Ser model system were chosen to represent coffee MRPs. The reasons behind this decision were: (1) the α dicarbonyl compounds identified in Ara Ser model MRPs were the same as those identified in coffee extracts. (2) The antioxidant activity and reducing power of MRPs derived from Ara Ser system were significantly higher compared to MRPs recovered from Suc Ser model system. (3) The antioxidant activity of MRPs derived from Ara Ser MRPs extract was similar to that of coffee extract. Crude extracts derived from Ara Ser model system were further fractionated based on molecular weight and four fractions were obtained. 116 3.3.8 Chemical characteristics and antioxidant activity Ara-Ser model MRPs fractions 3.3.8.1Recovery of fractions derived from Ara Ser model system Multiple step ultrafiltration was conducted to separate MRPs recovered from Ara Ser model system into different molecular weight (MW) fractions, as reported earlier (e.g. Fraction I (MW>10KDa), Fraction II (1KDa (0.5KDa contained the highest recovery of MRPs in both LR and DR processed Ara Ser reactants (Table 3.14). Table 3.14 Recovery of Ara Ser MRPs ultrafiltraton fractions 1 Fractions (MW) 2 Recovery (% of crude MRPs extract) LR DR I (>10KDa) 24.0+3.5 b 22.7+3.3 b II (1 10KDa) 7.7+1.4 ax 16.5+4.1 aby III (0.5 1KDa) 21.3+3.2 by 12.5+2.6 ax IV (<0.5 KDa) 41.8+3.8 c 44.3+6.3 c 1 Values are expressed as mean + SD, n=3. abc represent significantly different means in each column; xy represent significantly different means in rows. Statistical analyses using two way ANOVA with Bonferroni post tests; statistical level of confidence set as 0.05. 2 Fractions were derived from Ara Ser MRPs extracts, based on molecular weight (MW). 3.3.8.2 Colorimetric measurements and browning of Ara Ser model MRPs Table 3.15 presents data on the lightness (L) and browning of ultrafiltration fractions recovered from Ara Ser MR system. Fractions I, II and III recovered from Ara Ser model MRPs showed greater darkness than Fractions IV (p<0.05).There was a pattern of relative increase in browning that corresponded to an increase in molecular weight in the four fractions recovered from Ara Ser MRPs. 117 Table 3.15 Lightness (L) and browning of fractions derived from Ara Ser model MR system 1 Fractions (MW) 2 L Browning 3 LR DR LR DR I (>10KDa) 20.9+0.1 a 21.0+3.1 a 0.98+0.06 c 1.20+0.29 d II (1 10KDa) 22.9+1.8 a 25.0+1.4 ab 0.92+0.07 c 0.96+0.07 c III (0.5 1KDa) 27.4+4.8 a 31.9+2.9 b 0.50+0.09 b 0.47+0.05 b IV (<0.5 KDa) 47.0+4.5 b 42.2+8.8 c 0.12+0.02 a 0.18+0.06 a 1 LR and DR represent light roasted and dark roasted MRPs. Values represent mean + SD, n = 3. abcd The means with different superscript letters in each column are significantly different. (p<0.05, two way ANOVA with Bonferroni post tests). 2 Fractions are derived from Ara Ser MRPs extracts, based on molecular weight (MW). 3 Browning intensities are absorbance reading at 420 nm. Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 3.3.8.3 Fluoresence and UV spectra Figure 3.14 shows the different fluorescence intensities obtained for crude extract and ultrafiltration fractions derived from Ara Ser MRPs, respectively. Maximum fluorescence intensity was found at 480 nm for Fractions I and II; 470 nm for Fraction III, and 460 nm for Fraction IV. Among the four ultrafiltration fractions derived from Ara Ser MRPs extract, Fraction III gave the highest fluorescence intensity (p<0.05). The peak at 400 nm emission wavelength that was presented in the spectra for the LR Ara Ser MRPs crude extract was not observed in any of the four fractions recovered from the Ara Ser crude extract. Figure 3.15 shows the different UV spectra obtained for Ara Ser model MRPs crude extract, as well as recovered ultrafiltration fractions. The UV spectral absorbance of Ara Ser Fractions I, II and III were found to continuously decrease as the wavelength increased. Fraction IV recovered from Ara Ser MRPs had a peak UV absorbance at 285 nm. 118 Figure 3.14 Fluorescence emission spectra (350 550 nm) of light roasted (LR ) and dark roasted (DR ) MRPs extracts and fractions. Ara Ser MRPs crude extracts (A), Fraction I (B), Fraction II (C), Fraction III (D) and Fraction IV(E), measured with the excitation wavelength set at 400 nm. Fractions were derived from Ara Ser MRPs extracts, based on molecular weight (MW). Samples were diluted to 0.25 mg/ml with Milli Q water prior to spectrum measurement. 119 Figure 3.15 Comparison of the UV spectra of light roasted (LR ) and dark roasted (DR ) MRPs extracts and fractions. Ara Ser MRPs crude extracts (A), Fraction I (B), Fraction II (C), Fraction III (D) and Fraction IV(E), measured over a range of absorbance wavelengthes (250 700 nm). Fractions were derived from Ara Ser MRPs extracts, based on molecular weight (MW). Samples were diluted to 0.5 mg/ml with distilled de ionized water prior to spectrum measurement. 120 3.3.8.4 Antioxidant activity of Ara Ser model MRPs fractions Table 3.16 summarizes the antioxidant activity measured on different ultrafiltration fractions collected from the model Ara Ser MRPs extracts. Fractions I and III recovered from both the LR and DR Ara Ser MRPs, possessed higher ORAC antioxidant activity compared to Fractions II and IV (p<0.05). Higher radical scavenging activity was observed in Fractions I and II for both LR and DR Ara Ser MRPs when using the TEAC assay. No significant difference in RP activity was found between all fractions tested (data in Appendix Table 9). Table 3.16 Antioxidant activity of fractionated Ara Ser MRPs 1 Fractions 2 Assays 3 ORAC TEAC LR DR LR DR I (>10 KDa) 1.48+0.05 c 1.46+0.09 c 0.88+0.07 c 0.88+0.03 c II (1 10 KDa) 0.87+0.03 b 0.88+0.01 b 0.80+0.06 c 0.83+0.06 c III (0.5 1KDa) 1.54+0.04 c 1.51+0.15 c 0.49+0.05 b 0.47+0.04 b IV (<0.5 KDa) 0.39+0.03 a 0.52+0.05 a 0.20+0.02 a 0.24+0.06 a 1 LR and DR represent light roasted and dark roasted model MRPs. Value represents mean + SD (n=3). abc The means with different superscript letters in each column are significantly different (p<0.05); no significant difference between LR and DR. Significant differences were analyzed using two way ANOVA with Bonferroni post tests. 2 Fractions were derived from Ara Ser MRPs extracts, based on molecular weight (MW). 3 ORAC represents oxygen radical absorption capacity; TEAC represents Trolox equivalent antioxidant capacity. ORAC and TEAC values are expressed as mmol Trolox equivalents/g freeze dried MRPs samples. 3.3.10 Cell-based bioactivity of Ara-Ser MRPs 3.3.10.1 Caco 2 MTT response MRPs recovered from heat processed Ara Ser reactants to generate model MRPs similar to that recovered from roasted coffee were found to reduce the viability of Caco 2 cells in both a concentration and time dependent manner. The concentration response curves of 121 Caco 2 cells after exposure to both LR and DR model MRP treatments are shown in Figure 3.16. Calculated IC50 values for cells treated with these model MRPs at different time periods are presented in Table 3.17. The result showed that a concentration of 1.0 mg/ml, similar to the concentrations of coffee samples used in experiment IIa, should be chosen for further cell based antioxidant experiments in order to compare the results to the coffee MRP data. It should be point out that LR and DR MRP at a concentration of 1.0 mg/ml treatment of Caco 2 cells for 72 h reduced cell viability by about 25%. This observed cytotoxicity was significant greater (p<0.05) compared to the MRP free control. 122 Figure 3.15 Effects of light roasted (LR) and dark roasted (DR) Ara Ser MRPs extracts on the tetrazolium reduction rate in the MTT assay after 3 h ( ), 12 h ( ), 24 h ( ), 48 h ( ) and 72 h ( ) incubation. A: LR; B: DR. Data (% control) represent means + SD obtained for triplicate samples measured in triplicate. 123 1 Table 3.17 IC 50 values of Ara Ser MRP extracts on Caco 2 cells using MTT assay IC 50 (mg/ml) LR DR 3h 13.72+0.59 d 14.20+0.84 e 12h 13.85+0.99 dy 9.41+0.23 dx 24h 10.26+0.24 cy 6.44+0.36 cx 48h 6.40+0.22 by 4.90+0.14 bx 72h 3.41+0.34 ay 1.94+0.14 ax 1 IC 50 is equal to the concentration of coffee that was required to reduce cell viability to half that of control in the MTT assay. Values represent means + SD obtained for triplicate samples measured in triplicate. abcde represent means in columns that are significantly different; xy represent significantly different means in rows. Statistical analyses were done by two way ANOVA with Bonferroni post tests; level of confidence set at 0.05. 3.3.10.2 Caco 2 cellular glutathione content A significant (p<0.05) reduction of GSH content was observed in Caco 2 cells after a 24h treatment with LR and DR Ara Ser MRPs, respectively (Figure 3.16). No significant change in GSH content was seen after relatively early (3 h) treatment, or after prolonged (72 h) treatments. The in vitro cytotoxic effect of MRPs on Caco 2 cells was not associated with an intracellular reduction of GSH content in the present study, since 24 h exposure of cells to 1.0 mg/ml MRPs did not correspond to a significant increase in cytotoxicity. 3.3.10.3 Caco 2 antioxidant enzyme activity Exposing Caco 2 cells to 1.0 mg/ml LR and DR Ara Ser model MRPs, respectively, did not result in significant changes in the activity for CAT and GR up to 72 h (data in Appendix Table 10 11). Reduced cellular GPX activity was observed after 3 h of exposure of Caco 2 cells to both LR and DR MRPs treatments (Figure 3.17). There was a trend for a decrease in Caco 2 SOD activity when cells were treated with LR and DR MRPs, and the effect was significant after 72 h of treatment (Figure 3.18). 124 Figure 3.16 Effect of Ara Ser MRPs extracts on glutathione (GSH) content in Caco 2 cells. Cells were exposed to1.0 mg/ml green light roasted (LR) and dark roasted (DR) MRPs for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. * indicates significant difference compared to corresponding control (p<0.05). Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. 125 Figure 3.17 Effect of Ara Ser MRPs extracts on glutathione peroxidase (GPX) activity in Caco 2 cells. Cells were exposed to 1.0 mg/ml light roasted (LR) and dark roasted (DR) MRPs for 3 h (A), 24 h (B) and 72 h (C). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. * indicates significant difference compared to corresponding control (p<0.05). Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. 126 Figure 3.18 Effect of Ara Ser MRPs extracts on superoxide dismutase (SOD) activity in Caco 2 cells. Cells were exposed to1.0 mg/ml light roasted (LR) and dark roasted (DR) MRPs for 3 h (a), 24 h (b) and 72 h (c). Cells incubated in culture medium were used as control. Results are mean + SD, n=3. * indicates significant difference compared to corresponding control (p<0.05). Statistical analysis was done by one way ANOVA with Dunnett’s multiple comparison tests. 127 3.3.11 Biological effects of Ara-Ser MRPs fractions on Caco-2 cells 3.3.11.1 Caco 2 MTT response Table 3.18 summarizes the MTT response of Caco 2 cells exposed to 1.0 mg/ml crude extract and Fractions II and III recovered from LR Ara Ser MR system, respectively. Caco 2 cells treated with MRPs crude extract for 3 h showed no significant changes in cell viability. However, Caco 2 cells incubated with Fraction III for 3 h produced a significant (p<0.05) increase in cell viability, which contrasted the reduced cell viability observed in Fraction II treated cells (p<0.05). Pretreatment of Caco 2 cells with 1.0 mg/ml MRPs fraction III for 3 h prevented a H2O2 induced reduction in cell MTT response, thus maintaining cell viability in a range that was common for non stressed control cells. A similar pretreatment of Caco 2 cells with crude extract and Fractions II failed to show protective effects. There was no significant change in Caco 2 cell viability after 24 h treatment of crude MRPs extract and Fractions II and III, respectively. Moreover, the reduced cell viability induced by H 2O2 was not prevented by pretreatment of cells with MRPs extract or related fractions for 24 h. 1 Table 3.18 Caco 2 MTT response to MRPs with (+) and without ( ) H 2O2 treatment MRPs 3h Treatment 24h Treatment H 2O2 + H 2O2 H 2O2 + H 2O2 Control 100+8 80+4* 100+6 80+4* Crude 103+2 73+3 88+2 71+4 Fraction II 82+4* 70+5 98+4 75+4 Fraction III 121+4* 101+2** 96+1 82+1 1 Data (% control) represent means + SD obtained for triplicate samples measured in triplicate. Cells treated with MRPs samples alone were compared to negative control ( H2O2), and cells pretreated with MRPs samples and further challenged by H2O2 were compared to positive control (+H2O2). * indicates significant difference in comparison ** with negative control ( H2O2), and indicates significant difference in comparison with positive control (+H2O2). Statistical analyses were done by one way ANOVA with Dunnett’s tests (p<0.05). 128 3.3.11.2 Caco 2 cellular glutathione content Treating Caco 2 cells with 5 mM H 2O2 for 2 h reduced the cellular GSH content by 16%, which was significantly lower (p<0.05) from the non stressed, negative control. The exposure of Caco 2 cells to the crude extract, as well as to Fractions II and III, derived from the LR Ara Ser MR system, also significantly (p<0.05) reduced the cellular GSH content (Figure 3.19). There was no significant difference in GSH content between cells pretreated with MRPs and the positive control (+H2O2). Figure 3.19 Effect of MRPs extracts and associated fractions derived from light roasted (LR) Ara Ser MR system on Caco 2 cellular glutathione (GSH) contents after 24h of treatment. Without ( ) H2O2: ; with (+) H2O2 : . GSH content in cells that were treated with MRPs samples alone was compared to that of negative control ( H2O2), and GSH content in cells that were pretreated with MRPs samples and further challenged by H2O2 was compared to that of control (+H2O2). * indicated significant difference in comparison with corresponding control (p<0.05, one way ANOVA with Dunnett’s tests). 3.3.11.3 Caco 2 antioxidant enzyme activities The antioxidant enzyme activities of Caco 2 cells after a 24 h treatment with MRPs crude extract and related Fractions derived from LR Ara Ser model system are presented in Figure 3.20. Moreover, the same treatments with added H 2O2 induced stress are also shown in Figure 3.20. No significant change in GPX activity was observed in Caco 2 129 cells treated with Ara Ser MRPs crude extract, or when exposed to Fraction III recovered from the Ara Ser MR model system. In contrast, treatment of cells with Fraction II from the same crude extract resulted in a significant reduction in GPX activity (p<0.05). Treatment of cells with H2O2 alone also produced a significant increase (p<0.05) in Caco 2 GPX activity compared to the negative control ( H2O2). Cells pretreated with Fraction II recovered from LR Ara Ser MR system exhibited reduced (p<0.05) GPX activity after H2O2 treatment, compared to the positive control (+H2O2). Treating Caco 2 cells with H2O2 did not result in significant changes in cellular SOD activity. However, Caco 2 cells pretreated with Fraction II recovered from Ara Ser MRPs extract showed significantly reduced SOD activity after further H2O2 exposure (p<0.05). A significant (p<0.05) increase in CAT activity occurred in Caco 2 cells treated with H2O2. The activity of CAT was not significantly altered when cells were exposed to either the crude extract or the ultrafiltration fractions recovered from Ara Ser model system, respectively. Pretreatment of Caco 2 cells with MRPs crude extract did not prevent the increase in CAT activity caused by a H2O2 assault. However, Caco 2 cells pretreated with Fractions II and III recovered from MRPs crude extract had lower (p<0.05) CAT activity compared to the positive control (+H2O2). No significant change in Caco 2 GR activity was found between treatments and control cells (data in Appendix Table 12). 130 Figure 3.20 Effect of light roasted (LR) Ara Ser MRP extracts and fractions on Caco 2 cellular antioxidant enzyme activities after 24h of treatment. A: glutathione peroxidase (GPX); B: superoxide dismutase (SOD); C: catalase (CAT). Without ( ) H2O2: ; with (+) H2O2 : . Cells treated with MRPs samples alone were compared to negative control ( H2O2), and cells pretreated with MRPs samples and further challenged by H2O2 were compared to positive control (+H2O2). * indicates significant difference in comparison ** with negative control ( H2O2), and indicates significant difference in comparison with positive control (+H2O2). Statistical analyses were done by one way ANOVA with Dunnett’s tests (p<0.05). 131 3.3.12 Gene regulation of MRPs on the human oxidative stress and antioxidant defense system (HOSAD) in Caco-2 cells Results demonstrating the potential effect of MRPs derived from Ara Ser model systems on Caco 2 antioxidant gene regulation are presented in Table 3.19. After treatment of cells with the LR Ara Ser MRPs, significant changes in the expression of 10 genes associated with the HOSAD were observed using a PCR array (fold change>2, p<0.05). Of particular interest was the finding that expression of Caco 2 GPX2 , PRDX4 was up regulated 4.57 fold and 2.36 fold, respectively, whereas expression of iNOS was up regulated 2.90 fold. The expression of PRG3 was down regulated 8.21 fold, and SEPP1 was down regulated 3.11 fold. In contrast, Caco 2 cells treated with the MRPs derived from DR Ara Ser model system had 5 genes that were up regulated and no down regulation of genes. The expression of several genes that was altered in Caco 2 cells after treatment with coffee samples was also markedly changed in MRPs treated cells (Figure 3.21). 132 Table 3.19 Genes differently expressed in Caco 2 after incubation with Ara Ser MRPs 1 Gene symbol Treatment LR DR GPX2 +4.57 +8.79 SELS +2.41 n/c PRDX4 +2.36 n/c iNOS +2.09 +2.14 MPO +2.05 n/c PRG3 8.21 n/c PTGS1 4.28 n/c SEPP1 3.11 n/c PRNP 2.07 n/c MSRA 2.01 n/c ALOX12 n/c +4.42 ANGPTL7 n/c +4.05 CCS n/c +2.14 1 Data represent fold changes of differently expressed genes in treated cells compared to control cells (n=3, fold change>2, p<0.05). “+” = up regulation; “ ” = down regulation; n/c = no significant change or fold change<2. The name and function of these genes are described in Appendix (Table 8). 133 Figure 3.21 Antioxidant genes expression in Caco 2 cells treated with light roasted (LR: ), dark roasted (DR: ) Ara Ser MRPs compared to those in control cells. A: up regulated gene; B: down regulated gene. ** indicates p<0.01, * indicates p<0.05. GPX2 = glutathione peroxidase 2; SELS = Selenoprotein S; PRDX4 = peroxiredoxin 4; iNOS = inducible nitric oxide synthase; PRG3 = proteoglycan 3; PTGS1 = protaglandin endoperoxide synthase 1; SEPP1 = selenoprotein P; MSRA = methionine sulfoxide reductase A. The name and function of these genes are described in Appendix (Table 8). 134 3.4 Discussion 3.4.1 Chemical characteristics of coffee extracts and model MRPs The many challenges associated with the isolation and identification of individual products of the Maillard reaction derived from coffee has prevented a clear understanding of the structure activity relationships between coffee MRPs and antioxidant and bioactive potential, respectively. In Experiment IIa of the present study, non roasted, green coffee beans were used as a negative control with the understanding that this food matrix, albeit containing bioactive phenolics, such as CGA and caffeine, did not contain products of the Maillard Reaction. Model MRPs recovered from heated Ara Ser reactants were used in Experiment IIb as a reference for characterizing the bioactivity of MRPs generated during coffee roasting. The formation of MRPs and the degradation of phenolics in roasted coffee beans were characterized by a battery of fluorescence, UV vis spectra and tri stimulus color measurements made on non roasted and roasted coffee beans, as well as the model MRPs. From the fluorescence spectra, it could be ascertained that phenolics, particularly CGA were involved in the formation of both high molecular weight and low molecular weight MRPs during coffee roasting process. This finding agrees with results obtained in the previous chapter and also from a previous study, that showed CGA present in GB were incorporated into the high molecular weight melanoidin polymers (MW>10KDa) (Bekedam et al. , 2008d). It is also likely that high molecular weight phenolics, such as tannins (Savolainen, 1992) and phenolic glycosides (Clifford, 1985), were retained in the coffee fraction that contained the high molecular weight melanoidins (MW>10KDa). However, there is no indication that these polyphenolic compounds were incorporated 135 into the melanoidin structures (Clifford, 1985; Savolainen, 1992). Since no correlation could be made between the changes in fluorescence and molecular weight of derived model MRPs after roasting, it remains uncertain if the generation of high molecular weight MRPs can be explained simply by the polymerization reactivity of low molecular weight MRPs. Making a comparison between the UV vis spectra of the high molecular weight fractions derived from the Ara Ser model system and coffee extracts, respectively, showed some degree of similarity in the chromophoric sub structures that could be attributed to melanoidin formation in both the model, and the food system. A previous study has reported similar UV vis spectra between melanoidins derived from glucose casein model system and those isolated from dark beer (Hofmann, 1998b). The results therefore support, and extend the hypothesis that melanoidins derived from sugar amino acid, or sugar protein MR model systems, can be used to model the more complex food systems that contain many more discrete chromophores (Figure 4.1) (Hofmann, 1998b). The basic melanoidin skeletons from different MR sources will likely depend on the type of sugar, amino acids and proteins that are involved in the reaction. However, the results herein indicate that some similarities in the chromophoric sub structures or chromophoric low molecular MRPs indeed exist, and thus confirm the cross linking expected to occur between amino acid residues that form the high molecular weight melanoidins. 136 Figure 4.1 UV vis spectrum of a typical melanoidin ( ) and of individual chromophoric sub structures ( ) (Hofmann, 1998b). The Hunter L a b color scale and the absorbance readings taken at 420 nm have also been used previously to monitor the development of colored compounds during Maillard reaction (Kitts and Hu, 2005; Gokmen and Senyuva, 2006). However, these methods were found to be insensitive in this study for showing detectable colorimetric differences between LR and DR in both coffee extracts and the model MRPs. The results however, do show that there was a positive correlation between the darkness and browning, with increased molecular weight of the constituents recovered in the individual fractions. The degree of browning was found to be related more to the type of sugar than the degree of roasting, which agrees with other findings from different thermally processed food matrices, or model MRPs (Kurosaki et al. , 1989; Lingnert, 1990; Jing and Kitts, 2002). 137 3.4.2 Antioxidant activity and reducing power of coffee constituents 3.4.2.1 The underlying antioxidant mechanisms of MRPs derived from coffee extracts It has been shown in the previous studies that coffee brews exhibit antioxidant activity in both the ORAC and TEAC assays (Pellegrini et al. , 2003; del Castillo et al. , 2005; Delgado Andrade et al. , 2005; Sanchez Gonzalez et al. , 2005), which agrees with the findings in the present study. However, the concentration of phenolic non Maillard antioxidants in coffee brews was relatively high in some studies due to the mild roasting condition employed. In other studies, the phenolic content or the roasting conditions were not provided and therefore results of antioxidant activity could not be explained completely. It is therefore difficult to gain insight into the antioxidant property associated with coffee MRPs due to interference of phenolics and the complicity of the reaction products. The roasting conditions used for the commercial LR and DR coffee beans in the present study were more severe compared to those used in other studies (del Castillo et al. , 2002; del Castillo et al. , 2005; Sacchetti et al. , 2009). In this study, the plant phenolic antioxidants were mostly degraded during the roasting process, which was demonstrated from the CGA absorbance spectra derived from the coffee extracts. Other workers have also reported 84 to 95% loss of CGA in coffee extracts prepared using the same coffee bean species and similar roasting conditions and brewing methods (Trugo, 1984; Budryn et al. , 2009). Therefore, with this in mind, it is most likely that the antioxidant activity of the coffee extracts and related fractions reported in the present study are depended mostly on the presence of coffee MRPs. Furthermore, the present results also demonstrate that MRPs derived from coffee extracts exhibit peroxyl radicals scavenging activity in the ORAC assay by a transfer of hydrogen atoms to oxygen radicals, also referred to as the 138 HAT (hydrogen atom transfer) antioxidant reaction mechanism (Mayer, 2004). Similar antioxidant capacity of coffee MRPs also involves the transfer of single electron (SET mechanism) to oxidants, which was shown herein by the TEAC and Gu’s RP assays, which reduces the ABTS radicals and the ferric ions, respectively (Mayer, 2004). 3.4.2.2 Antioxidant activity of phenolic constituents in green coffee extracts and MRPs derived from roasted coffee extracts Despite several reports on the antioxidant activity of the roasted coffee brews/extracts, the antioxidant capacity of coffee extracts derived from green, unroasted coffee beans, in particular, has not been well studied. Results from the present study showed that GB extracts had higher antioxidant activity compared to roasted coffee extracts, which agrees with previous findings (Daglia et al. , 2000; Gomez Ruiz et al. , 2008). Chlorogenic acids monoester, 5 CQA, was suggested to possess the highest antioxidant activity among the different CGA esters present in GB in scavenging hydroxyl and peroxyl radicals (Daglia et al. , 2000; Daglia et al. , 2004). The results from the present study indicate, however, that CGA diesters and mixed esters have higher peroxyl radical scavenging activity compared to CGA monoesters (CGA diesters mostly present in Fraction III of GB extract and monoester mostly present in Fraction IV of GB extract). There are few studies that have reported on the antioxidant activity of CGA diesters and mixed esters, albeit the chemical structures are well known (Figure 4.2) (Farah and Donangelo, 2006). 139 A B C D Figure 4.2 Chlorogenic acids and related compounds according to chemical characteristics. (A) Basic compounds: CA: caffeic acid; FA: ferulic acid; pCoA: p coumaric acid; (B) monoesters of quinic acid with hydroxy cinnamic acids: caffeoylquinic acids (CQA), with 3 isomers (3 , 4 and 5 CQA); (C) di esters of quinic acid with caffeic acid: dicaffeoylquinic acids (diCQA), with 3 isomers (3,4 diCQA; 3,5 diCQA; 4,5 diCQA); and (D) mixed esters: six mixed diesters of caffeoylferuloyl quinic acids (CFAQ). (Farah and Donangelo, 2006) The antioxidant activity of MRPs derived from roasted coffee extracts is relatively lower compared to that of the CGA present in the GB extract, based on the data from the current study. This result extents the finding from a previous study, where a lower antioxidant activity of coffee melanoidins compared to CGA was reported (Delgado 140 Andrade et al. , 2005). Nevertheless, the antioxidant activity of coffee extract is still higher than that of green tea and black tea extracts (Cao et al. , 1996), and coffee MRPs have been mentioned to be great contributors to the total dietary antioxidant intake (Svilaas et al. , 2004). In agreement with the results reported in Chapter II and also a previous study (Somoza et al. , 2003), results herein showed that low molecular weight (MW<1KDa) MRPs derived from roasted coffee contribute to the most of the total ORAC, and TEAC antioxidant activity as well as the reducing power of the whole coffee extracts. This result is partially due to a greater proportion of compounds present in the low molecular weight fractions that exhibit antioxidant capacity, compared to compounds that make up the high molecular weight fractions (MW>1KDa) recovered from the coffee extracts. Our results also indicate that no positive or negative correlation exists between the antioxidant activity of coffee MRPs and the molecular weight of the compounds in these fractions. In addition, coffee MRPs showing the highest antioxidant activity in scavenging peroxyl radicals, expressed by an underlying HAT mechanism, did not have the highest ABTS radicals scavenging activity which occurs by a SET mechanism. In a recent study, Gu et al. , reported that MRPs of different molecular weights derived from casein glucose MR model system exhibited distinctly different antioxidant activities that included scavenging free radicals, sequestering metal ions and inhibiting lipid oxidation (Gu et al. , 2009). These results indicate that MRPs could act simultaneously with different mechanisms of action. 141 3.4.2.3 ORAC, TEAC and Gu’s RP assays assessing the antioxidant activity of coffee As mentioned, the ORAC assay measures the antioxidant activities of chain breaking antioxidants against peroxyl radicals, based on the HAT mechanism (Cao et al. , 1993; Ou et al. , 2001; Mayer, 2004). Compared to the ORAC assay used in other studies that test the sample of a single concentration (Cao et al. 1993; Ou et al. 2001), the improved ORAC assay by Kitts and Hu measures the peroxyl radical quenching capacity of an antioxidant over a range of different concentrations and calculates the ORAC value by dividing the slope of the regression equation of the sample by the slope of Trolox calibrator (Kitts and Hu, 2005; Saenz et al. , 2009). This modification offers a greater sensitivity and precision of measurement and produces consistent results for assessing the peroxyl radical scavenging activity of coffee extracts in the present study. Different Trolox equivalents (TE) units have been used to express the ORAC value of coffee, including TE/liter of coffee brew, TE/gram of freeze dried coffee extract, and TE/gram of coffee beans. These differences make the comparison of ORAC antioxidant activity of coffee brews/extracts between studies difficult. The use of Trolox equivalents/ gram of coffee extract (dry matter basis) represents a measure of antioxidant activity from coffee constituents that is independent of the difference in the concentration of coffee brews/extracts, where there is the use of a Trolox calibrator. The measurement of the SET specific antioxidant action, which also reflects the biological function of the antioxidant is relatively difficult to achieve compared to assessing the HAT mechanism (Ou et al. , 2002). With the TEAC assay, measuring the ABTS radical scavenging activity, and FRAP assay (ferric reducing antioxidant power), or Gu’s RP assay that measures the total reducing power of the antioxidant, simple and 142 very common used methods were popular for assessing antioxidant activity of dietary components (Karakaya et al. , 2001; Pellegrini et al. , 2003). The results of the current study indicate that Gu’s RP assay is a relatively insensitive method compared to the TEAC assay in assessing the SET activity of MRPs derived from the coffee extract. In addition, reducing power measurement estimates only the ferric reducing activity, and is not necessarily relevant to the total antioxidant capacity. Therefore, we suggest that ORAC and TEAC assays are preferable methods to evaluate the antioxidant activity of MRPs derived from coffee extract. Additional assays that have been used to measure • antioxidant capacity against other ROS, such as O 2 , ONOO , and singlet oxygen, have not been employed to measure the total antioxidant activity of coffee and MRPs derived from coffee extracts against multiple sources of ROS. 3.4.2 Biological effects of green coffee, roasted coffee and model MRPs on the antioxidant defense system in Caco-2 cells 3.4.2.1 MRPs derived from coffee and MR model systems inhibit the activity of antioxidant enzymes in Caco 2 cells Despite the strong in vitro antioxidant activity of MRPs derived from either complex food systems or simple model reaction systems, supporting data on the antioxidant effect of MRPs from in vivo studies are mostly inconsistent. Feeding rats a MRP containing diet has been shown to increase the total antioxidant capacity in plasma (Somoza et al. , 2005). Slightly increased plasma content of GSH was reported in humans that consumed five cups of coffee per day for a week (Esposito et al. , 2003). In contrast, a recent study has reported no changes in serum biomarkers of oxidative stress and antioxidant defenses (e.g. 143 hydroperoxides content, CAT, SOD and GPX) after coffee consumption (Seiquer et al. , 2008). In another study, an increased oxidative stress associated with coffee consumption has been reported (Sakamoto et al. , 2003). It is safe to conclude that the in vivo antioxidant mechanisms associated with MRPs are not fully understood, nor have they been demonstrated in human subjects. In the present study, treating Caco 2 cells with MRPs extracts derived from coffee and MR model systems resulted in a reduced cellular GSH content that corresponded to reduced activities of specific antioxidant enzymes. The only exception of this was an increased cellular GPX activity after 24 h coffee incubation. A similar result has been reported in a previous study that incubated HepG2 cells with coffee melanoidins showing a reduced cellular GSH content (Goya et al. , 2007). In addition, treatment of human lymphocytes and Int 407 cells with MRPs derived from different MR model systems has been reported to result in a reduced cellular GSH content and lower CAT, GR, GPX, and SOD activities (Yen et al. , 2002; Jing and Kitts, 2004b). At present, the mechanism for the inhibitory effect of MRPs on the activity of specific antioxidant enzymes remains unknown; however, several hypotheses have been suggested. Antioxidant enzymes require metal cofactors in order to reach maximum efficiency. For example, SOD consists of proteins co factored with copper, zinc, or manganese (Harris, 1992). Iron is also required as a cofactor for CAT (Tanswell and Freeman, 1984; Minotti and Aust, 1992). The metal sequestering activity of melanoidins has been shown for copper (Wijewickreme et al. , 1997; Wijewickreme and Kitts, 1998a) and iron (Morales et al. , 2005), two cofactors for SOD and CAT activity, respectively. It is therefore plausible that part of the inhibitory effect of MRPs, particularly the high 144 molecular weight melanoidins, could be related to metal sequestering activities (Wijewickreme et al. , 1997; Wijewickreme and Kitts, 1998a, b; Morales et al. , 2005). However, the mechanism for the enzyme inhibitory action associated with low molecular weight MRPs appears to be different from melanoidins, since the metal chelating properties of low molecular weight MRPs are limited (Delgado Andrade and Morales, 2005). It is also known that enzymes containing a functional group, such as the heme active site for CAT, require a catalytic three dimensional form in order to be reactive (Putnam et al. , 2000). Binding to these enzymes in either the active site or the non active site can reduce the activity to react with the substrate (Wyvratt and Patchett, 1985; Putnam et al. , 2000). A recent study has reported that methylglyoxal, a product of MR, can exhibit an inhibitory effect on GPX activity by binding to the GSH binding site and inactivate GPX in a time and dose dependent manner (Park et al. , 2003). Other α dicarbonyl MRPs, such as 3 deoxyglucosone, glyoxal and phenylglyoxal have also been reported to inactivate GPX (Park et al. , 2003). In addition, methylglyoxal has been found to inhibit the activity of SOD, GST, and CAT, and reduce GSH content in both in vitro and in vivo studies (Choudhary et al. , 1997). These particular α dicarbonyls were recovered in both coffee and model MRPs. Thus, the potential of α dicarbonyl MRPs to act as enzyme inhibitors, competing with the substrate for the active center of the specific antioxidant enzymes, and lowering the antioxidant enzyme activity, could explain the similar reduced enzyme activities for GPX, CAT and SOD observed for both coffee and model derived MRPs. 145 3.4.2.2 Effect of melanoidins on cellular oxidative stress induced by different oxidants In the present study, pretreatment of Caco 2 cells with MRP extract and the high molecular weight melanoidin fraction, respectively, derived from coffee and model systems showed no protection against H 2O2 induced cellular oxidative stress. Similar findings have been reported with model MRPs having no protection on DNA damage in human lymphocytes induced by H 2O2 (Yen et al. , 2002). In contrast, one study reported a protective effect of coffee melanoidins against tert butylhydroperoxide ( t BOOH) induced damage in HepG2 cells (Goya et al. , 2007). These results indicate that the protective effect of MRPs could be dependent on the source of the prooxidant that was used to induce oxidative cell damage. t BOOH , a well know oxidizing agent, leads to a loss of CAT activity and a subsequent increase in GPX and GR activity that reflects cell compensation to the loss of CAT against hydrogen peroxide and other peroxides (Pichorner et al. , 1993; Goya et al. , 2007). In the presence of a HAT agent, t BOOH is converted to t butanol, which is a non reactive substrate (Pichorner et al. , 1993). As discussed in the previous section, MRPs possess strong antioxidant activity by donating hydrogen atoms (HAT mechanism), which can explain the protective effect against t BOOH induced changes in the cellular antioxidant defense reported by Goya et al. (2007). This result contrasts the findings of the present study that showed no affinity for MRPs to scavenge H 2O2. In fact, several researches have also reported MRPs derived from both Maillard model systems and food systems (e.g. coffee) actually produce H 2O2 as a by product of the reaction (Namiki and Hayashi, 1975; Nagao et al. , 1986; Roberts and Lloyd, 1997; Yen et al. , 2002; Muscat et al. , 2007; Hegele et al. , 2009). It is important to also note that H 2O2 production from MRPs has been reported to increase in 146 the cell culture medium and result in cytotoxicity of HepG2 cells (Hegele et al. 2009). In the present study, exposing Caco2 cells to H 2O2, following a pretreatment with MRPs provided no protection and moreover actually increased cytotoxicity. This result may be explained by the fact that the amount of H 2O2 generated from the presence of MRPs in culture medium exposed to cells, along with the actual addition of H 2O2, collectively contributed to the observed Caco 2 cytotoxicity. The results of this study also showed that a greater cytotoxic response to MRPs was attributed to both the crude extract and the high molecular weight (MW>1KDa) fraction, but not to the low molecular weight fractions. Therefore, it is possible that this difference between high molecular weight and low molecular weight MRPs reflects that affinity of high molecular weight melanoidins to generate H 2O2. 3.4.3 Coffee and the expression of Redox-sensitive genes in Caco-2 cells 3.4.3.1 Coffee constituents and regulation of antioxidant gene expression in Caco 2 cells Data from the present study indicate that coffee extracts contain potential signaling molecules that can both up and down regulate the expression of specific genes involved in the oxidative stress and antioxidant defense systems in Caco 2 cells. Extracts derived from non roasted coffee beans showed little effect on the antioxidant gene expression in Caco 2 cells, which indicates that caffeine and natural phenolics are unlikely to be the main signaling molecules. Treating Caco 2 cells with coffee extracts resulted in similar changes in the expression of specific antioxidant genes as treating Caco 2 cells with H 2O2. This observation indicates that H 2O2 is involved in the regulation of antioxidant gene expression by coffee. The finding of H 2O2 as a signaling molecule in coffee induced gene 147 regulation process also agrees with a previous study, which showed that coffee induced the activation and nuclear translocation of the transcription factor NF кB (nuclear factor kappa B) via the generation of H 2O2 (Muscat et al. , 2007). In addition, MRPs may also be involved in Caco 2 cell gene regulation after coffee treatment, as demonstrated by the result that the expression of some genes (e.g. MBL2 , SELS , PRG3 , and MSRA ) was changed in coffee and model MRPs treated cells, but not in H 2O2 treated cells. The potential of coffee to regulate antioxidant gene expression has been reported in previous studies, where coffee induced the expression of genes that contain antioxidant response elements (ARE) (Cavin et al. , 2008; Higgins et al. , 2008). Coffee diterpenes, cafestol and kahweol (C+K), were reported to be responsible for the specific antioxidant/chemopreventive gene regulatory effects associated with coffee (Cavin et al. , 2002; Higgins et al. , 2008). However, it is important to remark that significant levels of C+K are only found in some special coffee preparations, such as Turkish and boiled coffees (Gross et al. , 1997). In most coffee brews, for example, the filtered coffee that was used in the present study, the concentration of diterpenes would not be an issue and therefore not attributed to a biological response (Gross et al. , 1997; Cavin et al. , 2008). In general, data from the current study indicates that specific MRPs present in the different coffee extracts, in addition to the possible generation of the H 2O2 in the cell culture system, collectively produced the gene specific effects observed in the PCR array. This is the first time the expression of a panel of antioxidant genes in a cell line exposed to coffee has been reported. 148 3.4.3.2 Coffee induced the expression of specific ARE driven antioxidant genes In the present study, coffee induced the expression of GPX2 (gastrointestinal glutathione peroxidase), SRXN1 (sulfiredoxin 1), TXNRD1 (thioredoxin reductase 1), PRDX1 (peroxiredoxin 1), PRDX4 (peroxiredoxin 4) and PRDX6 (peroxiredoxin 6) in Caco 2 cells. These genes all contain antioxidant response elements (ARE) in the promoters, which are sensitive to oxidative stress (Lee et al. , 2003; Singh et al. , 2006; Soriano et al. , 2008; Chowdhury et al. , 2009). In contrast, the expression of CAT (catalase), which also contains ARE sequence in the promoter, was down regulated in Caco 2 cells after coffee treatment. This result indicates that the induction of ARE driven genes in Caco 2 cells by coffee is likely selective and specific. It has been reported that catalase inhibits the activation of ARE driven genes (Lee et al. , 2001; Holland et al. , 2009). Therefore, it is possible that the down regulation of CAT expression in Caco 2 cells by coffee may facilitate the induction of specific ARE driven genes. The induction of other ARE driven genes by coffee has been reported previously, which include the genes for NQO1, UGT1A6, HO 1, AKR7A1, GSTP1, GSTA1, GSTA3, GSTA4, GSTA5, and GCLC (Cavin et al. , 2008; Higgins et al. , 2008). The induction of ARE driven genes by coffee was also shown to be concentration dependent and cell type specific (Higgins et al. , 2008). It has been suggested that altering the expression of ARE driven genes is a key molecular mechanism for detoxification and chemopreventive effects (Hayes et al. , 1999). For example, glutathione peroxidase and peroxiredoxin are important for cellular defense against H 2O2 (Sies, 1993; Rhee et al. , 2005) . These peroxidases use GSH and/or thioredoxin as electron donor for detoxification of H 2O2 (Sies, 1993; Fisher et al. , 1999; Manevich and Fisher, 2005). It is also well known that glutathione peroxidase is the 149 primary defense against H 2O2 at high concentrations (Masaki et al. , 1998; Wijeratne et al. , 2005). Oxidized GSH and thioredoxin are converted to reduced forms by glutathione reductase and thioredoxin reductase (TXNRD1). In the present study, the expression of TXNRD1 was induced in Caco 2 cells after coffee treatment. However, the mRNAs of glutathione reductase and glutathione synthetase were not induced by coffee. This specific induction of glutathione peroxidase and peroxiredoxin, but not glutathione reductase and glutathione synthetase in Caco 2 cells after coffee treatment, corresponds to the reduced cellular glutathione content observed in Caco 2 cells after coffee treatment. These results indicate that coffee treatment of cells could induce oxidative stress, which leads to the induction of specific antioxidant genes. The overall effect of coffee treatment may be an increased resistance to oxidative damage by maintaining a certain level of oxidative stress in cells. In fact, it has been suggested in other studies that the molecular mechanism of an antioxidant action involves the generation of ROS as second messengers for antioxidant gene induction (Favreau and Pickett, 1991; Li and Jaiswal, 1994; Pinkus et al. , 1996; Yang et al. , 2006; Holland et al. , 2009). The transcription factor Nrf2 (Nuclear factor erythroid 2 p45 subunit related factor 2) has been implicated to be the central protein that interacts with the ARE to activate ARE driven gene transcription constitutively, or in response to an oxidative stress signal (Nguyen et al. , 2003; Jaiswal, 2004). Data from the present study indicate that coffee induced oxidative stress, particularly the generation of H 2O2 in the cell culture system (Hegele et al. , 2009), may function as a signal that activates Nrf2 dependent induction of ARE driven gene expression. Indeed, H 2O2 treatment of rat cardiomyocytes has been reported to cause a rapid increase in the translation of Nrf2 protein (Purdom Dickinson et 150 al. , 2007). The regulation of Nrf2/ARE signaling pathway by coffee constituents, C+K and caffeine, has been addressed in previous studies (Cavin et al. , 2002; Okano et al. , 2008). C+K disrupt the cytoplasmic Keap1 Nrf2 complex through thiol modification of cysteine residues in Keap1, therefore releasing Nrf2 and permitting its translocation to nucleus, where Nrf2 transcriptionally activates ARE driven genes (Dinkova Kostova et al. , 2002). Caffeine activates MAPK/ERK signal pathway so as to phosphorylate Nrf2 and permits its translocation to the nucleus, where Nrf2 activates ARE driven genes (Okano et al. , 2008). Taken together, these reported results indicate that coffee extracts may induce Nrf2/ARE signaling pathway by increasing the translation and nuclear translocation of Nrf2, which involves different coffee constituents. 3.4.3.3 Coffee and iNOS induction Coffee treatment of Caco 2 cells induced the mRNA of iNOS (inducible nitric oxide synthase), which produces nitric oxide, a major endogenous modulator with diverse biological actions (Nussler and Billiar, 1993; Yetik Anacak and Catravas, 2006; Chen and Kitts, 2008a). The expression of iNOS in Caco 2 cells can be induced by certain cytokines, such as interleukin 1 (IL 1) and tumor necrosis factor (TNF) (Lowenstein et al. , 1992). A recent study demonstrated that oxidative stress can also lead to the up regulation of iNOS as well as eNOS (endothelial nitric oxide synthase) in vivo (Zhen et al. , 2008). The proposed mechanism of oxidative stress induced up regulation of iNOS is mainly through the activation of redox sensitive transcription factor, NF кB (Xie et al. , 1994; Liu et al. , 1997; Zhen et al. , 2008). Notably, it has been reported that coffee and model MRPs extracts led to nuclear translocation of NF кB in macrophages via the generation of H 2O2 (Muscat et al. , 2007). Hydrogen peroxide has been suggested to be 151 the primary activator of NF кB signaling pathway (Muscat et al. , 2007; Zhen et al. , 2008). In addition to one NF кB binding site, the promoter region of iNOS gene contains two binding sites for activator protein (AP) 1, which is also important for the transcriptional induction of iNOS (Chu et al. , 1998). It has been reported in a previous study that caffeine induced the expression of AP 1 protein (Svenningsson et al. , 1995). Thus, these data suggest that coffee induce the expression of iNOS through activation of AP 1 and NF кB. Caffeine may augment or synergize H 2O2 induced expression of iNOS , which is supported by the observation of greater induction of iNOS in coffee treated cells compare to cells treated with H 2O2 and model MRPs, respectively. 3.4.3.4 Proposed pathway of hydrogen peroxide generation in coffee and Maillard reaction systems The potential of coffee and MRPs to generate H 2O2, especially in the cell culture system, is intriguing. High levels of hydrogen peroxide can cause oxidative stress, which is an important cause of cell damage. However, it has also been suggested that H 2O2 triggers different signaling pathways in a concentration dependent manner and this results in redox mediated changes in the function of enzymes, which include antioxidant enzymes and other proteins that protect cells against injury from ROS (Babior, 1997; Sablina et al. , 2005; Bossis and Melchior, 2006). Based on the current understanding of the schemes of the Maillard reaction and the autoxidation of monosaccharide and polyphenols (Wolff and Dean, 1987; Elgawish et al. , 1996; Mochizuki et al. , 2002), the mechanism of how • ROS, including O 2 and H 2O2 are formed in coffee brews is proposed here for the first time (Figure 4.3). The general hypothesis is that many coffee constituents contain a special enediol group or an enediol like chemical group (Figure 4.3A), which can 152 • generate superoxide anions (O 2 ) when in the presence of trace metal catalysis (Figure 4.3, Scheme I). The basic scheme I is based on the available mechanisms of the formation of superoxide anions from Amadori products, hydroxyaldehyde, and polyphenols, which are all coffee components (Wolff and Dean, 1987; Elgawish et al. , 1996; Yaylayan et al. , 1998; Nakanishi et al. , 2001; Akagawa et al. , 2003). The initiation of Scheme I reaction is a metal catalysed process (Wolff and Dean 1987; Elgawish et al. 1996; Akagawa et al. 2003), which forms a very unstable resonance structure containing a delocalized electron system. This electron when transferred to an oxygen molecule will form a superoxide anion. Superoxide anions generated are spontaneously converted to H 2O2 through dismutation reaction according to Scheme II (Figure 4.3) (Babior, 1997; Akagawa et al. , 2003). Data from the present study and former studies indicate that hydrogen peroxide formation is associated with melanoidins. Previous studies have demonstrated that free radicals, including superoxide radicals are generated through sugar fragmentation and Amadori rearrangment during the early stage of the Maillard reaction (Namiki and Hayashi, 1983; Kawakishi et al. , 1994; Roberts and Lloyd, 1997). Both sugar fragments and Amadori products have been identified in the melanoidins structures (Cämmerer and Kroh, 1995). Phenolics have also been detected in melanoidins derived from coffee brews (Bekedam et al. , 2008d). These structural components of melanoidins all in common have the affinity to generate superoxide radicals and H 2O2, especially in phosphate buffer, which provides the optimal pH and also contains trace metal ions (Thornalley et al. , 1984; Kawakishi et al. , 1994; Akagawa et al. , 2003). The fact that melanoidins chelate metal ions may also favor the structure components generating H 2O2. 153 A. Enediol group OH OH R1 CC R2 Scheme I: the basic scheme - OH OH OH O O O O O n+ (n 1)+ M M O2 O2 R1 CC R2 R1 CC R2 R1 CC R2 R1 CC R 2 i. Amadori product CH NH R2 CH NH R2 2 2 CH2NH2R2 2 2 CH2NHR2 n n+ M(n 1)+ O O o C OH C OH M C O 2 2 ati C O iz ol en C OH C O 3 C O C O 2, CH2NH2R2 R1 R1 R1 R1 C O HO C H 1, 2 R1 en o HC NHR2 HC HC NR2 HC NR2 liz NR2 at (n 1)+ io Mn+ M O O2 n C OH C OH C O 2 C O HO C H C O C O C O R1 R1 R1 R1 ii. Hydroxyaldehyde (n 1)+ O O OH O - Mn+ M O O OH O O2 O2 R1 CC H R1 CC H R1 CC H R1 CC H dicarbonyl H iii. Phenolics OH OH n+ M(n 1)+ O O M O2 O2 R OH R O R O R O Scheme II + 2O2 + 2H H2O2 + O2 O H O + 2 + 2H2O 2 2 2OH Figure 4.3 Proposed pathway of H 2O2 formation in coffee brews. 154 CHAPTER IV GENERAL DISCUSSION AND CONCLUSIONS 155 4.1 GENERAL DISCUSSION 4.1.1 Chemical characteristics and antioxidant properties of coffee The development of brown color during the roasting process of coffee beans is directly related to the Maillard reaction. The UV, fluorescence and color measurements were used to characterize and compare the chemical properties of MRPs derived from coffee and MR model systems. A positive correlation between darkness and browning, with increased molecular weight of MRPs was observed. In contrast, the fluorescent property of MRPs did not show the same correlation with the molecular weight. No positive, or negative, correlation was observed between the antioxidant activities of MRPs and the degree of browning or the fluorescent intensity. Results from previous studies regarding the relationship between the antioxidant activity of MRPs and color and fluorescent properties are inconsistent (Monti et al. , 1999; Morales and Jimenez Perez, 2001; Murakami et al. , 2002; Chen and Kitts, 2008b). The discrepancies could due to different reactants, different reaction conditions, and different assays that were used to assess the antioxidant activity. However, taken together with the results from the present study, it can be conclude that color and fluorescent properties are useful indicators for the development of Maillard reaction, but are not directly correlated to the antioxidant potential of crude or fractionated MRPs. By extension, it can also be concluded that chromophore sub structures of MRPs derived from coffee and model systems are not responsible for antioxidant action, which agrees with a previous finding (Rufian Henares and Morales, 2007c). 156 4.1.2 Coffee, antioxidant enzymes and antioxidant genes The results of the chemical antioxidant assays clearly showed that coffee has high antioxidant activity by scavenging free radicals and also possesses reducing activity. However, potential interaction of bioactive components in coffee and living cells resulted in different antioxidant mechanisms of actions. In fact, a prooxidant property associated with coffee was observed in Caco 2 cells, which involved a reduced cellular glutathione status after coffee treatment. Experimental evidence indicates that antioxidant defense systems can act synergistically or antagonistically, according to the level or activities of the systems involved (Chow, 1988). Coffee treatment of Caco 2 cells enhanced the non enzymatic antioxidant defense, which may compensate for the negative effects on the activities of antioxidant enzymes. In addition, hydrogen peroxide generated in the cell culture system as a result of coffee exposure is capable of signaling and communicating critical information to activate specific signaling pathways, which will influence various cellular processes that lead to improved cell functions depending on the concentration of H 2O2 (Scandalios, 2005; Veal et al. , 2007). In the present study, the induction of ARE drive gene expression in Caco 2 cells exposed to coffee extract indicated an enhanced cellular antioxidant defense system. Results from the present study also indicate that coffee could regulate gene expressions in Caco 2 cells through Nrf2, NF κB and Ap 1 signaling pathways. Many cell functions, such as inflammatory response and anti carcinogenic effects, are mediated either directly or indirectly through these same pathways (Tak and Firestein, 2001; Zhang and Gordon, 2004). In general, Caco 2 cells adapted to the coffee added environment by altering the 157 expression of specific antioxidant and oxidative stress response genes, which are likely to result in a more responsive defense system when challenged by ROS. 158 4.2 CONCLUSION Maillard reaction products were shown to be the prevailing antioxidants present in roasted coffee extracts. Chromophore sub structures of the coffee MRPs are not responsible for the antioxidant action. No positive, or negative, correlation exists between the antioxidant activity of coffee MRPs and molecular weight. The mechanisms of the antioxidant action associated with coffee MRPs involve both the hydrogen atom transfer (HAT) mechanism and single electron transfer (SET) mechanism. Reducing power is relatively less important to predicting antioxidant capacity of MRPs. Treatment of Caco 2 cells with MRPs derived from both coffee extracts and model systems resulted in increased cellular oxidative stress, as evidenced by a reduced glutathione content that corresponded to modified activities of specific antioxidant enzymes, namely GPX, CAT and SOD. The inhibitory effect of coffee extracts on specific antioxidant enzymes is suggested to be related to the metal sequestering activity of high molecular weight MRPs and the presence of α dicarbonyl MRPs, which are potential enzyme inhibitors. In addition, exposing Caco 2 cells to roasted coffee extracts resulted in many changes in the expression of genes that are involved in human oxidative stress and antioxidant defense system. The induction of ARE driven gene by coffee resulted in an increased endogenous defense mechanisms that could respond by enhanced protection against various types of environmental stresses. The induction of ARE driven genes by coffee is suggested herein to be associated with the Nrf2 signaling pathway. The specific induction of iNOS gene expression in Caco 2 cells also indicates a possible role for coffee intake in inflammatory and immune responses. Results indicated that the 159 activation of iNOS gene by coffee is likely to be mediated by the activation of the transcription factors, AP 1 and NF κB. Hydrogen peroxide generated in the cell culture system as a consequence of coffee exposure, may serve as a signaling molecule that is involved in the gene regulatory effect associated with coffee extracts. The generation of H2O2 by coffee constituents was proposed. In addition, our data for the first time showed the antioxidant character of coffee MRPs on a molecular level. The overall gene regulatory effect of coffee constituents on Caco 2 cells is more likely to be the combined effects of all bioactive constituents, which involves a complex interplay of chemical and biological reactions and responses. 160 4.3 SUGGESTIONS FOR FUTURE RESEARCH (1) To investigate the specific effect of α dicarbonyl MRPs, for example, methylglyoxal on the expression of antioxidant genes in Caco 2 cells; the combined effect of methylglyoxal and hydrogen peroxide on the activities of antioxidant enzymes and the expression of antioxidant genes. 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Cells were exposed to 0.1 mg/ml light roasted (LR) and dark roasted (DR) coffee for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. Table 2 Effect of coffee extracts on the glutathione reductase activity in Caco 2 cells 1 3 h 24 h 72 h Control 20+3 17+1 19+1 LR 21+2 19+2 21+2 DR 23+2 18+0 18+2 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as mU/mg protein. Cells were exposed to 0.1 mg/ml light roasted (LR) and dark roasted (DR) coffee for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. Table 3 Effect of coffee extracts on the superoxide dismutase activity in Caco 2 cells 1 3 h 24 h 72 h Control 5.1+0.2 5.0+0.3 4.8+0.2 LR 6.1+0.5 4.1+0.0 4.8+0.8 DR 6.0+0.6 4.3+0.6 5.1+0.5 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as U/mg protein. Cells were exposed to 0.1 mg/ml light roasted (LR) and dark roasted (DR) coffee for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. Table 4 Effect of coffee extracts on the glutathione reductase activity in Caco 2 cells 1 3 h 24 h 72 h Control 17+1 18+2 19+4 GB 16+2 20+2 19+4 LR 16+1 15+1 16+2 DR 16+1 18+2 18+5 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as mU/mg protein. Cells were exposed to 1.0 mg/ml green coffee bean extract (GB), light roasted (LR) and dark roasted (DR) coffee for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. 185 Table 5 Effect of coffee extracts on the superoxide dismutase activity in Caco 2 cells 1 3 h 24 h 72 h Control 5.9+0.9 5.9+0.5 6.4+1.1 GB 5.7+0.3 5.7+0.2 6.1+0.4 LR 5.8+0.1 5.0+0.6 5.5+0.2 DR 5.6+0.4 5.0+0.3 5.7+0.4 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as U/mg protein. Cells were exposed to 1.0 mg/ml green coffee bean extract (GB), light roasted (LR) and dark roasted (DR) coffee for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. Table 6 Effect of light roasted coffee extracts and fractions on Caco 2 superoxide 1 dismutase activity with (+) or without ( ) H 2O2 treatment H 2O2 +H 2O2 Control 5.9+0.5 5.4+0.6 Defatted 5.0+0.6 5.6+0.3 Fraction II 5.0+0.4 4.8+0.3 Fraction III 5.3+0.6 6.2+0.6 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as U/mg protein. Cells were exposed to 1.0 mg/ml light roasted coffee samples in culture medium for 24 h. Some cells were further treated with H 2O2 for 2 h. Cells incubated in culture medium were used as positive control. Cells treated with H2O2 added culture medium were used as positive control. Table 7 Effect of green coffee bean extracts and fractions on Caco 2 superoxide 1 dismutase activity with (+) or without ( ) H 2O2 treatment H 2O2 +H 2O2 Control 5.9+0.5 5.4+0.6 Defatted 5.7+0.2 5.3+0.2 Fraction II 5.3+0.4 5.1+0.1 Fraction III 6.0+0.6 5.6+0.3 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as U/mg protein. Cells were exposed to 1.0 mg/ml green coffee bean extracts and fractions in culture medium for 24 h. Some cells were further treated with H 2O2 for 2 h. Cells incubated in culture medium were used as negative control. Cells treated with H 2O2 added culture medium were used as positive control. 186 Table 8 Description of genes that are differently expressed in Caco 2 cells upon incubation with coffee and MRPs samples Gene Description Functional classification ALB Albumin Good binding capacity. Regulates the colloidal osmotic pressure of blood ALOX12 Arachidonate 12 Oxygenase and 14,15 leukotriene A4 synthase lipoxygenase activity AOX1 Aldehyde oxidase 1 Produce H2O2 and under certain conditions, can catalyze the formation of superoxide. ATOX1 antioxidant protein 1 Could bind and deliver cytosolic copper to the homolog copper ATPase proteins. May be important in cellular antioxidant defense. CAT Catalase Protect cells from the toxic effects of H 2O2. CCS Copper chaperone for Delivers copper to copper zinc superoxide dismutase SOD (SOD) GPX2 Glutathione peroxidase Responsible for the majority of the glutathione 2 (gastrointestinal) dependent H 2O2 reducing activity in the epithelium of the gastrointestinal tract. Could play a major role in protecting mammals from the toxicity of ingested organic hydroperoxides. iNOS inducible Nitric oxide Produces nitric oxide (NO) which is a messenger synthase molecule with diverse functions throughout the body. MBL2 Mannose binding lectin Is capable of host defense against pathogens by (protein C) 2 activiating the classical complement pathway independently of the antibody. MPO Myeloperoxidase Part of the host defense system of polymorphonuclear leukocyte. MSRA Methionine sulfoxide Has an important function as a repair enzyme for reductase A proteins that have been inactivated by oxidation. MT3 Metallothionein 3 Binds heavy metals. Could play unique roles in homeostasis of the central nervous system and in the etiology of neuropathological disorders. Protective effect on DNA damage in response to reactive oxygen species (ROS). NCF2 Neutrophil cytosolic Required for activation of the latent NADPH factor 2 oxidase NME5 Non metastatic cells 5, Confers protection from cell death and alters the protein expressed in cellular levels of several antioxidant enzymes. May (nucleoside diphosphate play a role in kinase) spermiogenesis by increasing the ability of late stage spermatids to eliminate ROS. NOX5 NADPH oxidase, EF Generate superoxide and functions as a H+ channel hand calcium binding in a Ca 2+ –dependent manner. domain 5 187 Table 8 Description of genes that are differently expressed in Caco 2 cells upon incubation with coffee and MRPs samples (continued) Gene Description Functional classification PRDX1 Peroxiredoxin 1 Reduce hydrogen peroxide and alkyl hydroperoxides PRDX4 Peroxiredoxin 4 Involved in redox regulation of the cell. Regulates the activation of NF кB in the cytosol by a modulation of IкB α phosphorylation. PRDX6 Peroxiredoxin 6 Involved in redox regulation of the cell. Reduce H2O2 and short chain organic, fatty acid, and phospholipids hydroperoxides. Play a role in the regulation of phospholipid turnover as well as in protection against oxidative injury. PREX1 Phosphatidylinositol Functions as a RAC guanine nucleotide exchange 3,4,5 trisphosphate factor (GEF). dependent Rac exchange factor 1 PRG3 Proteoglycan 3 p53 responsive gene. Possesses cytotoxic and cytostimulatory activities. PRNP Prion protein Response to oxidative stress. Cellular copper ion homeostasis PTGS1 Prostaglandin May play an important role in regulating or endoperoxide synthase 1 promoting cell proliferation. (prostaglandin G/H synthase and cyclooxygenase) PXDN Peroxidasin homolog p53 responsive gene SELS Selenoprotein S Involved in the degradation process of misfolded endoplasmic reticulum (ER) luminal proteins. SEPP1 Selenoprotein P, p53 responsive gene. Possesses cytotoxic and plasma,1 cytostimulatory activities. SRXN1 Sulfiredoxin 1 homolog Contributes to oxidative stress resistance by reducing cysteine sulfinic acid formed under exposure to oxidants in the peroxiredoxins PRDX1, PRDX2, PRDX3 and PRDX4. May catalyze the reduction in a multi step process by acting both as a specific phosphotransferase and a thioltransferase TXNRD1 Thioredoxin reductase 1 May possess glutaredoxin activity as well as thioredoxin reductase activity. 188 Table 9 Reducing power of Ara Ser MRPs fractions 1 Sample LR DR Fraction I 0.24+0.01 a 0.25+0.01 a Fraction II 0.22+0.02 a 0.24+0.03 a Fraction III 0.23+0.02 a 0.26+0.02 a Fraction IV 0.19+0.02 a 0.23+0.03 a 1 Reducing power (RP) values (mg CGA/g freeze dried samples) are the mean ± SD obtained for triplicate solutions measured in triplicate. LR = light roast; DR = dark roast. Table 10 Effect of Ara Ser MRPs on the catalase activity in Caco 2 cells 1 3 h 24 h 72 h Control 2.8+0.4 2.6+0.3 3.0+0.4 LR 2.2+0.3 2.0+0.6 2.8+0.4 DR 2.1+0.4 2.2+0.4 2.7+0.2 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as U/mg protein. Cells were exposed to 1.0 mg/ml light roasted (LR) and dark roasted (DR) MRPs in culture medium for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. Table 11 Effect of Ara Ser MRPs on the glutathione reductase activity in Caco 2 cells 1 3 h 24 h 72 h Control 17+1 18+2 19+4 LR 17+1 20+2 21+2 DR 17+1 18+2 19+2 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as mU/mg protein. Cells were exposed to 1.0 mg/ml light roasted (LR) and dark roasted (DR) MRPs in culture medium for 3 h, 24 h and 72 h. Cells incubated in culture medium were used as control. 189 Table 12 Effect of Ara Ser MRPs extracts and fractions on Caco 2 superoxide dismutase 1 activity with (+) or without ( ) H 2O2 treatment H 2O2 +H 2O2 Control 18+2 15+1 Defatted 20+2 17+3 Fraction II 17+3 15+1 Fraction III 20+1 15+1 1 The values (mean + SD, n = 3) represent enzyme activity, expressed as mU/mg protein. Cells were exposed to 1.0 mg/ml light roasted MRPs extracts and fractions in culture medium for 24 h. Some cells were further treated with H 2O2 for 2 h. Cells incubated in culture medium were used as negative control. Cells treated with H 2O2 added culture medium were used as positive control. 190